Mutations Involve Changes in the Base Sequence of DNA

We now turn from DNA replication to DNA mutations and repair. Several types of mutations are known: (1) the
substitution of one base pair for another, (2) the deletion of one or more base pairs, and (3) the insertion of one or more
base pairs. The spontaneous mutation rate of T4 phage is about 10-7 per base per replication. E. coli and Drosophila
melanogaster have much lower mutation rates, of the order of 10-10.
The substitution of one base pair for another is the a common type of mutation. Two types of substitutions are possible.
A transition is the replacement of one purine by the other or that of one pyrimidine by the other. In contrast, a
transversion is the replacement of a purine by a pyrimidine or that of a pyrimidine by a purine.
Watson and Crick suggested a mechanism for the spontaneous occurrence of transitions in a classic paper on the DNA
double helix. They noted that some of the hydrogen atoms on each of the four bases can change their location to produce
a tautomer. An amino group (-NH2) can tautomerize to an imino form ( NH). Likewise, a keto group (
can tautomerizeto an enol form
. The fraction of each type of base in the formof these imino and enol tautomers is about10-4. These transient tautomers
can form nonstandard base pairs that fit into a double helix. For example, the imino tautomer of adenine can pair with
cytosine .This A*-C pairing (the asterisk denotes the imino tautomer) would allow C to become
incorporated into a growing DNA strand where T was expected, and it would lead to a mutation if left uncorrected. In the
next round of replication, A* will probably retautomerize to the standard form, which pairs as usual with thymine, but
the cytosine residue will pair with guanine. Hence, one of the daughter DNA molecules will contain a G-C base pair in
place of the normal A-T base pair.
Tautomerization
The interconversion of two isomers that differ only in the position of
protons (and, often, double bonds).
Some Chemical Mutagens Are Quite Specific
Base analogs such as 5-bromouracil and 2-aminopurine can be incorporated into DNA and are even more likely than
normal nucleic acid bases to form transient tautomers that lead to transition mutations. 5-Bromouracil, an analog of
thymine, normally pairs with adenine. However, the proportion of 5-bromouracil in the enol tautomer is higher than that
of thymine because the bromine atom is more electronegative than is a methyl group on the C-5 atom. Thus, the
incorporation of 5-bromouracil is especially likely to cause altered base-pairing in a subsequent round of DNA
replication .Other mutagens act by chemically modifying the bases of DNA. For example, nitrous acid (HNO2) reacts with bases that
contain amino groups. Adenine is oxidatively deaminated to hypoxanthine, cytosine to uracil, and guanine to xanthine.
Hypoxanthine pairs with cytosine rather than with thymine .Uracil pairs with adenine rather than with
guanine. Xanthine, like guanine, pairs with cytosine. Consequently, nitrous acid causes A-T G-C transitions.
A different kind of mutation is produced by flat aromatic molecules such as the acridines .These
compounds intercalate in DNA that is, they slip in between adjacent base pairs in the DNA double helix.
Consequently, they lead to the insertion or deletion of one or more base pairs. The effect of such mutations is to alter the
reading frame in translation, unless an integral multiple of three base pairs is inserted or deleted. In fact, the analysis of
such mutants contributed greatly to the revelation of the triplet nature of the genetic code.
Some compounds are converted into highly active mutagens through the action of enzymes that normally play a role in
detoxification. A striking example is aflatoxin B1, a compound produced by molds that grows on peanuts and other
foods. A cytochrome P450 enzyme converts this compound into a highly reactive epoxide (Figure
27.45). This agent reacts with the N-7 atom of guanosine to form an adduct that frequently leads to a G-C-to-T-A
transversion.
27.6.2. Ultraviolet Light Produces Pyrimidine Dimers
The ultraviolet component of sunlight is a ubiquitous DNA-damaging agent. Its major effect is to covalently link
adjacent pyrimidine residues along a DNA strand .Such a pyrimidine dimer cannot fit into a double helix,
and so replication and gene expression are blocked until the lesion is removed.
A Variety of DNA-Repair Pathways Are Utilized
The maintenance of the integrity of the genetic message is key to life. Consequently, all cells possess mechanisms to
repair damaged DNA. Three types of repair pathways are direct repair, base-excision repair, and nucleotide-excision
repair .An example of direct repair is the photochemical cleavage of pyrimidine dimers. Nearly all cells contain a
photoreactivating enzyme called DNA photolyase. The E. coli enzyme, a 35-kd protein that contains bound N 5,N 10-
methenyltetrahydrofolate and flavin adenine dinucleotide cofactors, binds to the distorted region of DNA. The enzyme
uses light energy specifically, the absorption of a photon by the N 5,N 10-methenyltetrahydrofolate coenzyme to
form an excited state that cleaves the dimer into its original bases.
The excision of modified bases such as 3-methyladenine by the E. coli enzyme AlkA is an example of base-excision
repair. The binding of this enzyme to damaged DNA flips the affected base out of the DNA double helix and into the
active site of the enzyme .Base flipping also occurs in the enzymatic addition of methyl groups to DNA
bases .The enzyme then acts as a glycosylase, cleaving the glycosidic bond to release the damaged base.
At this stage, the DNA backbone is intact, but a base is missing. This hole is called an AP site because it is apurinic
(devoid of A or G) or apyrimidinic (devoid of C or T). An AP endonuclease recognizes this defect and nicks the
backbone adjacent to the missing base. Deoxyribose phosphodiesterase excises the residual deoxyribose phosphate unit,
and DNA polymerase I inserts an undamaged nucleotide, as dictated by the base on the undamaged complementary
strand. Finally, the repaired strand is sealed by DNA ligase.
One of the best-understood examples of nucleotide-excision repair is the excision of a pyrimidine dimer. Three
enzymatic activities are essential for this repair process in E. coli .First, an enzyme complex consisting of
the proteins encoded by the uvrABC genes detects the distortion produced by the pyrimidine dimer. A specific uvrABC
enzyme then cuts the damaged DNA strand at two sites, 8 nucleotides away from the dimer on the 5 side and 4
nucleotides away on the 3 side. The 12-residue oligonucleotide excised by this highly specific excinuclease (from the
Latin exci,"to cut out") then diffuses away. DNA polymerase I enters the gap to carry out repair synthesis. The 3 end of
the nicked strand is the primer, and the intact complementary strand is the template. Finally, the 3 end of the newly
synthesized stretch of DNA and the original part of the DNA chain are joined by DNA ligase.
The Presence of Thymine Instead of Uracil in DNA Permits the Repair of
Deaminated Cytosine
The presence in DNA of thymine rather than uracil was an enigma for many years. Both bases pair with adenine. The
only difference between them is a methyl group in thymine in place of the C-5 hydrogen atom in uracil. Why is a
methylated base employed in DNA and not in RNA? The existence of an active repair system to correct the deamination
of cytosine provides a convincing solution to this puzzle.
Cytosine in DNA spontaneously deaminates at a perceptible rate to form uracil. The deamination of cytosine is
potentially mutagenic because uracil pairs with adenine, and so one of the daughter strands will contain an U-A base pair
rather than the original C-G base pair .This mutation is prevented by a repair system that recognizes uracil
to be foreign to DNA. This enzyme, uracil DNA glycosylase, is homologous to AlkA. The enzyme hydrolyzes the
glycosidic bond between the uracil and deoxyribose moieties but does not attack thymine-containing nucleotides. The
AP site generated is repaired to reinsert cytosine. Thus, the methyl group on thymine is a tag that distinguishes thymine
from deaminated cytosine. If thymine were not used in DNA, uracil correctly in place would be indistinguishable from
uracil formed by deamination. The defect would persist unnoticed, and so a C-G base pair would necessarily be mutated
to U-A in one of the daughter DNA molecules. This mutation is prevented by a repair system that searches for uracil and
leaves thymine alone. Thymine is used instead of uracil in DNA to enhance the fidelity of the genetic message. In
contrast, RNA is not repaired, and so uracil is used in RNA because it is a less-expensive building block.

Many Cancers Are Caused by Defective Repair of DNA
cancers are caused by mutations in genes associated with growth control. Defects in
DNA-repair systems are expected to increase the overall frequency of mutations and, hence, the likelihood of a
cancer-causing mutation. Xeroderma pigmentosum, a rare human skin disease, is genetically transmitted as an autosomal
recessive trait. The skin in an affected homozygote is extremely sensitive to sunlight or ultraviolet light. In infancy,
severe changes in the skin become evident and worsen with time. The skin becomes dry, and there is a marked atrophy
of the dermis. Keratoses appear, the eyelids become scarred, and the cornea ulcerates. Skin cancer usually develops at
several sites. Many patients die before age 30 from metastases of these malignant skin tumors.
Ultraviolet light produces pyrimidine dimers in human DNA, as it does in E. coli DNA. Furthermore, the repair
mechanisms are similar. Studies of skin fibroblasts from patients with xeroderma pigmentosum have revealed a
biochemical defect in one form of this disease. In normal fibro-blasts, half the pyrimidine dimers produced by ultraviolet
radiation are excised in less than 24 hours. In contrast, almost no dimers are excised in this time interval in fibroblasts
derived from patients with xeroderma pigmentosum. The results of these studies show that xeroderma pigmentosum can
be produced by a defect in the excinuclease that hydrolyzes the DNA backbone near a pyrimidine dimer. The drastic
clinical consequences of this enzymatic defect emphasize the critical importance of DNA-repair processes. The disease
can also be caused by mutations in eight other genes for DNA repair, which attests to the complexity of repair processes.
Defects in other repair systems can increase the frequency of other tumors. For example, hereditary nonpolyposis
colorectal cancer (HNPCC, or Lynch syndrome) results from defective DNA mismatch repair. HNPCC is not rare as
many as 1 in 200 people will develop this form of cancer. Mutations in two genes, called hMSH2 and hMLH1, account
for most cases of this hereditary predisposition to cancer. The striking finding is that these genes encode the human
counterparts of MutS and MutL of E. coli. The MutS protein binds to mismatched base pairs (e.g., G-T) in DNA. An
MutH protein, together with MutL, participates in cleaving one of the DNA strands in the vicinity of this mismatch to
initiate the repair process .It seems likely that mutations in hMSH2 and hMLH1 lead to the accumulation
of mutations throughout the genome. In time, genes important in controlling cell proliferation become altered, resulting
in the onset of cancer.
Some Genetic Diseases Are Caused by the Expansion of Repeats of Three
Nucleotides
Some genetic diseases are caused by the presence of DNA sequences that are inherently prone to errors in the
course of replication. A particularly important class of such diseases are characterized by the presence of long
tandem arrays of repeats of three nucleotides. An example is Hunt-ington disease, an autosomal dominant neurological
disorder with a variable age of onset. The mutated gene in this disease expresses a protein called huntingtin, which is
expressed in the brain and contains a stretch of consecutive glutamine residues. These glutamine residues are encoded by
a tandem array of CAG sequences within the gene. In unaffected persons, this array is between 6 and 31 repeats,
whereas, in those with the disease, the array is between 36 and 82 repeats or longer. Moreover, the array tends to become
longer from one generation to the next. The consequence is a phenomenon called anticipation: the children of an
affected parent tend to show symptoms of the disease at an earlier age than did the parent.
The tendency of these trinucleotide repeats to expand is explained by the formation of alternative structures in DNA
replication .Part of the array within the daughter strand can loop out without disrupting base-pairing
outside this region. DNA polymerase extends this strand through the remainder of the array, leading to an increase in the
number of copies of the trinucleotide sequence.
A number of other neurological diseases are characterized by expanding arrays of trinucleotide repeats. How do these
long stretches of repeated amino acids cause disease? For huntingtin, it appears that the polyglutamine stretches become
increasingly prone to aggregate as their length increases; the additional consequences of such aggregation are still under
active investigation.
Many Potential Carcinogens Can Be Detected by Their Mutagenic Action on
Bacteria
Many human cancers are caused by exposure to chemicals. These chemical carcinogens usually cause mutations, which
suggests that damage to DNA is a fundamental event in the origin of mutations and cancer. It is important to identify
these compounds and ascertain their potency so that human exposure to them can be minimized. Bruce Ames devised a
simple and sensitive test for detecting chemical mutagens. In the Ames test, a thin layer of agar containing about 109
bacteria of a specially constructed tester strain of Salmonella is placed on a petri dish. These bacteria are unable to grow
in the absence of histidine, because a mutation is present in one of the genes for the biosynthesis of this amino acid. The
addition of a chemical mutagen to the center of the plate results in many new mutations. A small proportion of them
reverse the original mutation, and histidine can be synthesized. These revertants multiply in the absence of an external
source of histidine and appear as discrete colonies after the plate has been incubated at 37°C for 2 days .For example, 0.5 g of 2-aminoanthracene gives 11,000 revertant colonies, compared with only 30 spontaneous
revertants in its absence. A series of concentrations of a chemical can be readily tested to generate a dose-response curve.
These curves are usually linear, which suggests that there is no threshold concentration for mutagenesis.
Some of the tester strains are responsive to base-pair substitutions, whereas others detect deletions or additions of base
pairs (frameshifts). The sensitivity of these specially designed strains has been enhanced by the genetic deletion of their
excision-repair systems. Potential mutagens enter the tester strains easily because the lipopolysaccharide barrier that
normally coats the surface of Salmonella is incomplete in these strains.
A key feature of this detection system is the inclusion of a mammalian liver homogenate .Recall that
some potential carcinogens such as aflatoxin are converted into their active forms by enzyme systems in the liver or
other mammalian tissues .Bacteria lack these enzymes, and so the test plate requires a few milligrams of
a liver homogenate to activate this group of mutagens.
The Salmonella test is extensively used to help evaluate the mutagenic and carcinogenic risks of a large number of
chemicals. This rapid and inexpensive bacterial assay for mutagenicity complements epidemiological surveys and animal
tests that are necessarily slower, more laborious, and far more expensive. The Salmonella test for mutagenicity is an
outgrowth of studies of gene-protein relations in bacteria. It is a striking example of how fundamental research in
molecular biology can lead directly to important advances in public health.

DNA Replication of Both Strands Proceeds Rapidly from Specific Start Sites

So far, we have met many of the key players in DNA replication. Here, we ask, Where on the DNA molecule does
replication begin, and how is the double helix manipulated to allow the simultaneous use of the two strands as templates?
In E. coli, DNA replication starts at a unique site within the entire 4.8 × 106 bp genome. This origin of replication, called
the oriC locus, is a 245-bp region that has several unusual features .The oriC locus contains four repeats
of a sequence that together act as a binding site for an initiation protein called dnaA. In addition, the locus contains a
tandem array of 13-bp sequences that are rich in A-T base pairs.
The binding of the dnaA protein to the four sites initiates an intricate sequence of steps leading to the unwinding of the
template DNA and the synthesis of a primer. Additional proteins join dnaA in this process. The dnaB protein is a
helicase that utilizes ATP hydrolysis to unwind the duplex. The single-stranded regions are trapped by a single-stranded
binding protein (SSB). The result of this process is the generation of a structure called the prepriming complex, which
makes single-stranded DNA accessible for other enzymes to begin synthesis of the complementary strands.
An RNA Primer Synthesized by Primase Enables DNA Synthesis to Begin
Even with the DNA template exposed, new DNA cannot be synthesized until a primer is constructed. Recall that all
known DNA polymerases require a primer with a free 3 -hydroxyl group for DNA synthesis. How is this primer formed?
An important clue came from the observation that RNA synthesis is essential for the initiation of DNA synthesis. In fact,
RNA primes the synthesis of DNA. A specialized RNA polymerase called primase joins the prepriming complex in a
multisubunit assembly called the primosome. Primase synthesizes a short stretch of RNA (~5 nucleotides) that is
complementary to one of the template DNA strands .The primer is RNA rather than DNA because DNA
polymerases cannot start chains de novo. Recall that, to ensure fidelity, DNA polymerase tests the correctness of the
preceding base pair before forming a new phosphodiester bond .RNA polymerases can start chains de
novo because they do not examine the preceding base pair. Consequently, their error rates are orders of magnitude as
high as those of DNA polymerases. The inge-nious solution is to start DNA synthesis with a low-fidelity stretch of
polynucleotide but mark it "temporary" by placing ribonucleotides in it. The RNA primer is removed by hydrolysis by a
5 3 exonuclease; in E. coli, the exonuclease is present as an additional domain of DNA polymerase I, rather than
being present in the Klenow fragment. Thus, the complete polymerase I has three distinct active sites: a 3 5
exonuclease proofreading activity, a polymerase activity, and a 5 3 exonuclease activity.
One Strand of DNA Is Made Continuously, Whereas the Other Strand Is
Synthesized in Fragments
Both strands of parental DNA serve as templates for the synthesis of new DNA. The site of DNA synthesis is called the
replication fork because the complex formed by the newly synthesized daughter strands arising from the parental duplex
resembles a two-pronged fork. Recall that the two strands are antiparallel; that is, they run in opposite directions. As
both daughter strands appear to grow in the same direction on cursory examination. However, all
known DNA polymerases synthesize DNA in the 5 3 direction but not in the 3 5 direction. How then does one
of the daughter DNA strands appear to grow in the 3 5 direction?
This dilemma was resolved by Reiji Okazaki, who found that a significant proportion of newly synthesized DNA exists
as small fragments. These units of about a thousand nucleotides (called Okazaki fragments) are present briefly in the
vicinity of the replication fork .As replication proceeds, these fragments become covalently joined
through the action of DNA ligase to form one of the daughter strands. The other new strand is
synthesized continuously. The strand formed from Okazaki fragments is termed the lagging strand, whereas the one
synthesized without interruption is the leading strand. Both the Okazaki fragments and the leading strand are synthesized
in the 5 3 direction. The discontinuous assembly of the lagging strand enables 5 3 polymerization at the
nucleotide level to give rise to overall growth in the 3 5 direction.
DNA Ligase Joins Ends of DNA in Duplex Regions
The joining of Okazaki fragments requires an enzyme that catalyzes the joining of the ends of two DNA chains. The
existence of circular DNA molecules also points to the existence of such an enzyme. In 1967, scientists in several
laboratories simultaneously discovered DNA ligase. This enzyme catalyzes the formation of a phosphodiester bond
between the 3 hydroxyl group at the end of one DNA chain and the 5 -phosphate group at the end of the other An energy source is required to drive this thermodynamically uphill reaction. In eukaryotes and archaea, ATP is
the energy source. In bacteria, NAD+ typically plays this role. We shall examine the mechanistic features that allow
these two molecules to power the joining of two DNA chains.
DNA ligase cannot link two molecules of single-stranded DNA or circularize single-stranded DNA. Rather, ligase seals
breaks in double-stranded DNA molecules. The enzyme from E. coli ordinarily forms a phosphodiester bridge only if
there are at least several base pairs near this link. Ligase encoded by T4 bacteriophage can link two blunt-ended doublehelical
fragments, a capability that is exploited in recombinant DNA technology.
Let us look at the mechanism of joining, which was elucidated by I. Robert Lehman donates its
activated AMP unit to DNA ligase to form a covalent enzyme-AMP (enzyme-adenylate) complex in which AMP is linked
to the -amino group of a lysine residue of the enzyme through a phosphoamide bond. Pyrophosphate is concomitantly
released. The activated AMP moiety is then transferred from the lysine residue to the phosphate group at the 5 terminus
of a DNA chain, forming a DNA-adenylate complex. The final step is a nucleophilic attack by the 3 hydroxyl group at
the other end of the DNA chain on this activated 5 phosphorus atom.
In bacteria, NAD+ instead of ATP functions as the AMP donor. NMN is released instead of pyrophosphate. Two hightransfer-
potential phosphoryl groups are spent in regenerating NAD+ from NMN and ATP when NAD+ is the adenylate
donor. Similarly, two high-transfer-potential phosphoryl groups are spent by the ATP-utilizing enzymes because the
pyrophosphate released is hydrolyzed. The results of structural studies revealed that the ATP- and NAD+-utilizing
enzymes are homologous even though this homology could not be deduced from their amino acid sequences alone.

DNA Replication Requires Highly Processive Polymerases
Enzyme activities must be highly coordinated to replicate entire genomes precisely and rapidly. A prime example is
provided by DNA polymerase III holoenzyme, the enzyme responsible for DNA replication in E. coli. The hallmarks of
this multisubunit assembly are its very high catalytic potency, fidelity, and processivity. Processivity refers to the ability
of an enzyme to catalyze many consecutive reactions without releasing its substrate. The holoenzyme catalyzes the
formation of many thousands of phosphodiester bonds before releasing its template, compared with only 20 for DNA
polymerase I. DNA polymerase III holoenzyme has evolved to grasp its template and not let go until the template has
been completely replicated. A second distinctive feature of the holoenzyme is its catalytic prowess: 1000 nucleotides are
added per second compared with only 10 per second for DNA polymerase I. This acceleration is accomplished with no
loss of accuracy. The greater catalytic prowess of polymerase III is largely due to its processivity; no time is lost in
repeatedly stepping on and off the template.
Processive enzyme
From the Latin procedere, "to go forward."
An enzyme that catalyzes multiple rounds of elongation or digestion
of a polymer while the polymer stays bound. A distributive enzyme,
in contrast, releases its polymeric substrate between successive
catalytic steps.
These striking features of DNA polymerase III do not come cheaply. The holoenzyme consists of 10 kinds of
polypeptide chains and has a mass of ~900 kd, nearly an order of magnitude as large as that of a single-chain DNA
polymerase, such as DNA polymerase I. This replication complex is an asymmetric dimer .The
holoenzyme is structured as a dimer to enable it to replicate both strands of parental DNA in the same place at the same
time. It is asymmetric because the leading and lagging strands are synthesized differently. A 2 subunit is associated
with one branch of the holoenzyme; 2 and ()2 are associated with the other. The core of each branch is the
same, an complex. The subunit is the polymerase, and the subunit is the proofreading 3 5 exonuclease.
Each core is catalytically active but not processive. Processivity is conferred by 2 and 2.
The source of the processivity was revealed by the determination of the three-dimensional structure of the 2 subunit
.This unit has the form of a star-shaped ring. A 35-Å-diameter hole in its center can readily accommodate
a duplex DNA molecule, yet leaves enough space between the DNA and the protein to allow rapid sliding and turning
during replication. A catalytic rate of 1000 nucleotides polymerized per second requires the sliding of 100 turns of
duplex DNA (a length of 3400 Å, or 0.34 m) through the central hole of 2 per second. Thus, 
2 plays a key role in
replication by serving as a sliding DNA clamp.
The Leading and Lagging Strands Are Synthesized in a Coordinated Fashion
The holoenzyme synthesizes the leading and lagging strands simultaneously at the replication fork .DNA
polymerase III begins the synthesis of the leading strand by using the RNA primer formed by primase. The duplex DNA
ahead of the polymerase is unwound by an ATP-driven helicase. Single-stranded binding protein again keeps the strands
separated so that both strands can serve as templates. The leading strand is synthesized continuously by polymerase III,
which does not release the template until replication has been completed. Topoisomerases II (DNA gyrase) concurrently
introduces right-handed (negative) supercoils to avert a topological crisis.
The mode of synthesis of the lagging strand is necessarily more complex. As mentioned earlier, the lagging strand is
synthesized in fragments so that 5 3 polymerization leads to overall growth in the 3 5 direction. A looping of
the template for the lagging strand places it in position for 5 3 polymerization .The looped laggingstrand
template passes through the polymerase site in one subunit of a dimeric polymerase III in the same direction as
that of the leading-strand template in the other subunit. DNA polymerase III lets go of the lagging-strand template after
adding about 1000 nucleotides. A new loop is then formed, and primase again synthesizes a short stretch of RNA primer
to initiate the formation of another Okazaki fragment.
The gaps between fragments of the nascent lagging strand are then filled by DNA polymerase I. This essential enzyme
also uses its 5 3 exonuclease activity to remove the RNA primer lying ahead of the polymerase site. The primer
cannot be erased by DNA polymerase III, because the enzyme lacks 5 3 editing capability. Finally, DNA ligase
connects the fragments.
DNA Synthesis Is More Complex in Eukaryotes Than in Prokaryotes
Replication in eukaryotes is mechanistically similar to replication in prokaryotes but is more challenging for a number of
reasons. One of them is sheer size: E. coli must replicate 4.8 million base pairs, whereas a human diploid cell must
replicate 6 billion base pairs. Second, the genetic information for E. coli is contained on 1 chromosome, whereas, in
human beings, 23 pairs of chromosomes must be replicated. Finally, whereas the E. coli chromosome is circular, human
chromosomes are linear. Unless countermeasures are taken, linear chromosomes are subject to
shortening with each round of replication.
The first two challenges are met by the use of multiple origins of replication, which are located between 30 and 300 kbp
apart. In human beings, replication requires about 30,000 origins of replication, with each chromosome containing
several hundred. Each origin of replication represents a replication unit, or replicon. The use of multiple origins of
replication requires mechanisms for ensuring that each sequence is replicated once and only once. The events of
eukaryotic DNA replication are linked to the eukaryotic cell cycle .In the cell cycle, the processes of DNA
synthesis and cell division (mitosis) are coordinated so that the replication of all DNA sequences is complete before the
cell progresses into the next phase of the cycle. This coordination requires several checkpoints that control the
progression along the cycle.
The origins of replication have not been well characterized in higher eukaryotes but, in yeast, the DNA sequence is
referred to as an autonomously replicating sequence (ARS) and is composed of an AT-rich region made up of discrete
sites. The ARS serves as a docking site for the origin of replication complex (ORC). The ORC is composed of six
proteins with an overall mass of ~400 kd. The ORC recruits other proteins to form the prereplication complex. Several of
the recruited proteins are called licensing factors because they permit the formation of the initiation complex. These
proteins serve to ensure that each replicon is replicated once and only once in a cell cycle. How is this regulation
achieved? After the licensing factors have established the initiation complex, these factors are marked for destruction by
the attachment of ubiquitin and subsequently destroyed by proteasomal digestion .DNA helicases separate the parental DNA strands, and the single strands are stabilized by the binding of replication
protein A, a single-stranded- DNA-binding protein. Replication begins with the binding of DNA polymerase , which is
the initiator polymerase. This enzyme has primase activity, used to synthesize RNA primers, as well as DNA polymerase
activity, although it possesses no exonuclease activity. After a stretch of about 20 deoxynucleotides have been added to
the primer, another replication protein, called protein replication factor C (RFC), displaces DNA polymerase and
attracts proliferating cell nuclear antigen (PCNA). Homologous to the 2 subunit of E. coli polymerase III, PCNA then
binds to DNA polymerase . The association of polymerase with PCNA renders the enzyme highly processive and
suitable for long stretches of replication. This process is called polymerase switching because polymerase has replaced
polymerase . Polymerase has 3 5 exonuclease activity and can thus edit the replicated DNA. Replication
continues in both directions from the origin of replication until adjacent replicons meet and fuse. RNA primers are
removed and the DNA fragments are ligated by DNA ligase.
Telomeres Are Unique Structures at the Ends of Linear Chromosomes
Whereas the genomes of essentially all prokaryotes are circular, the chromosomes of human beings and other eukaryotes
are linear. The free ends of linear DNA molecules introduce several complications that must be resolved by special
enzymes. In particular, it is difficult to fully replicate DNA ends, because polymerases act only in the 5 3 direction.
The lagging strand would have an incomplete 5 end after the removal of the RNA primer. Each round of replication
would further shorten the chromosome.
The first clue to how this problem is resolved came from sequence analyses of the ends of chromosomes, which are
called telomeres (from the Greek telos, "an end"). Telomeric DNA contains hundreds of tandem repeats of a
hexanucleotide sequence. One of the strands is G rich at the 3 end, and it is slightly longer than the other strand. In
human beings, the repeating G-rich sequence is AGGGTT.
The structure adopted by telomeres has been extensively investigated. Recent evidence suggests that they may form large
duplex loops .The single-stranded region at the very end of the structure has been proposed to loop back
to form a DNA duplex with another part of the repeated sequence, displacing a part of the original telomeric duplex. This
looplike structure is formed and stabilized by specific telomere-binding proteins. Such structures would nicely protect
and mask the end of the chromosome.
Telomeres Are Replicated by Telomerase, a Specialized Polymerase That
Carries Its Own RNA Template
How are the repeated sequences generated? An enzyme, termed telomerase, that executes this function has been purified
and characterized. When a primer ending in GGTT is added to the human enzyme in the presence of deoxynucleoside
triphosphates, the sequences GGTTAGGGTT and GGTTAGGGTTAGGGTT, as well as longer products, are generated.
Elizabeth Blackburn and Carol Greider discovered that the enzyme contains an RNA molecule that serves as the
template for elongation of the G-rich strand .Thus, the enzyme carries the information necessary to
generate the telomere sequences. The exact number of repeated sequences is not crucial.
Subsequently, a protein component of telomerases also was identified. From its amino acid sequence, this component is
clearly related to reverse transcriptases, enzymes first discovered in retroviruses that copy RNA into DNA. Thus,
telomerase is a specialized reverse transcriptase that carries its own template. Telomeres may play important roles in
cancer-cell biology and in cell aging.
III. Synthesizing the Molecules of Life 27. DNA Replication, Recombination, and Repair 27.4. DNA Replication of Both Strands Proceeds Rapidly from Specific Start Sites
III. Synthesizing the Molecules of Life 27. DNA Replication, Recombination, and Repair
Double-Stranded DNA Molecules with Similar Sequences Sometimes
Recombine
Most processes associated with DNA replication function to copy the genetic message as faithfully as possible.
However, several biochemical processes require the recombination of genetic material between two DNA molecules. In
genetic recombination, two daughter molecules are formed by the exchange of genetic material between two parent molecules.

1. In meiosis, the limited exchange of genetic material between paired chromosomes provides a simple mechanism for
generating genetic diversity in a population.
2. As we shall see in Chapter 33, recombination plays a crucial role in generating molecular diversity for antibodies and
some other molecules in the immune system.
3. Some viruses utilize recombination pathways to integrate their genetic material into the DNA of the host cell.
4. Recombination is used to manipulate genes in, for example, the generation of "gene knockout" mice .Recombination is most efficient between DNA sequences that are similar in sequence. Such processes are often referred
to as homologous recombination reactions.
Recombination Reactions Proceed Through Holliday Junction Intermediates
The Structural Insights module for this chapter shows how a recombinase
forms a Holliday junction from two DNA duplexes and suggests how this
intermediate is resolved to produce recombinants.
Enzymes called recombinases catalyze the exchange of genetic material that takes place in recombination. By what
pathway do these enzymes catalyze this exchange? An appealing scheme was proposed by Robin Holliday in 1964. A
key intermediate in this mechanism is a crosslike structure, known as a Holliday junction, formed by four polynucleotide
chains. Such intermediates have been characterized by a wide range of techniques including x-ray crystallography
.Note that such intermediates can form only when the nucleotide sequences of the two parental duplexes
are very similar or identical in the region of recombination because specific base pairs must form between the bases of
the two parental duplexes.
How are such intermediates formed from the parental duplexes and resolved to form products? Many details for this
process are now available, based largely on the results of studies of Cre recombinase from bacteriophage P1. This
mechanism begins with the recombinase binding to the DNA substrates . Four molecules of the enzyme
and their associated DNA molecules come together to form a recombination synapse. The reaction begins with the
cleavage of one strand from each duplex. The 5 -hydroxyl group of each cleaved strand remains free, whereas the 3 -
phosphoryl group becomes linked to a specific tyrosine residue in the recombinase. The free 5 ends invade the other
duplex in the synapse and attack the DNA-tyrosine units to form new phosphodiester-bonds and free the tyrosine
residues. These reactions result in the formation of a Holliday junction. This junction can then isomerize to form a
structure in which the polynucleotide chains in the center of the structure are reoriented. From this junction, the
processes of strand cleavage and phosphodiester-bond formation repeat. The result is a synapse containing the two
recombined duplexes. Dissociation of this complex generates the final recombined products.
Recombinases Are Evolutionarily Related to Topoisomerases
The intermediates that form in recombination reactions, with their tyrosine adducts possessing 3 -phosphoryl
groups, are reminiscent of the intermediates that form in the reactions catalyzed by topoisomerases. This
mechanistic similarity reflects deeper evolutionary relationships. Examination of the three-dimensional structures of
recombinases and type I topoisomerases reveals that these proteins are related by divergent evolution despite little amino
acid sequence similarity .From this perspective, the action of a recombinase can be viewed as an
intermolecular topoisomerase reaction. In each case, a tyrosine-DNA adduct is formed. In a topoisomerase reaction, this
adduct is resolved when the 5 -hydroxyl group of the same duplex attacks to reform the same phosphodiester bond that
was initially cleaved. In a recombinase reaction, the attacking 5 -hydroxyl group comes from a DNA chain that was not
initially linked to the phosphoryl group participating in the phosphodiester bond.

Double-Stranded DNA Can Wrap Around Itself to Form Supercoiled Structures

The separation of the two strands of DNA in replication requires the local unwinding of the double helix. This local
unwinding must lead either to the overwinding of surrounding regions of DNA or to supercoiling. To prevent the strain
induced by overwinding, a specialized set of enzymes is present to introduce supercoils that favor strand separation.
The Linking Number of DNA, a Topological Property, Determines the Degree of
Supercoiling
In 1963, Jerome Vinograd found that circular DNA from polyoma virus separated into two distinct species when it was
centrifuged. In pursuing this puzzle, he discovered an important property of circular DNA not possessed by linear DNA
with free ends. Consider a linear 260-bp DNA duplex in the B-DNA .Because the number of
residues per turn in an unstressed DNA molecule is 10.4, this linear DNA molecule has 25 (260/10.4) turns. The ends of
this helix can be joined to produce a relaxed circular DNA .A different circular DNA can be formed by
unwinding the linear duplex by two turns before joining its ends .What is the structural consequence of
unwinding before ligation? Two limiting conformations are possible: the DNA can either fold into a structure containing
23 turns of B helix and an unwound loop or adopt a supercoiled structure with 25 turns of B helix and 2
turns of right-handed (termed negative) superhelix .
Supercoiling markedly alters the overall form of DNA. A supercoiled DNA molecule is more compact than a relaxed
DNA molecule of the same length. Hence, supercoiled DNA moves faster than relaxed DNA when analyzed by
centrifugation or electrophoresis. The rapidly sedimenting DNA in Vinograd's experiment was supercoiled, whereas the
slowly sedimenting DNA was relaxed because one of its strands was nicked. Unwinding will cause supercoiling in both
circular DNA molecules and in DNA molecules that are constrained in closed configurations by other means.
Helical Twist and Superhelical Writhe Are Correlated with Each Other
Through the Linking Number
Our understanding of the conformation of DNA is enriched by concepts drawn from topology, a branch of mathematics
dealing with structural properties that are unchanged by deformations such as stretching and bending. A key topological
property of a circular DNA molecule is its linking number (Lk), which is equal to the number of times that a strand of
DNA winds in the right-handed direction around the helix axis when the axis is constrained to lie in a plane. For the
relaxed DNA shown in Figure 27.19B, Lk = 25. For the partly unwound molecule shown in part D and the supercoiled
one shown in part E, Lk = 23 because the linear duplex was unwound two complete turns before closure. Molecules
differing only in linking number are topological isomers (topoisomers) of one another. Topoisomers of DNA can be
interconverted only by cutting one or both DNA strands and then rejoining them.
The unwound DNA and supercoiled DNA and E are topologically identical but geometrically
different. They have the same value of Lk but differ in Tw (twist) and Wr (writhe). Although the rigorous definitions of
twist and writhe are complex, twist is a measure of the helical winding of the DNA strands around each other, whereas
writhe is a measure of the coiling of the axis of the double helix, which is called super-coiling. A right-handed coil is
assigned a negative number (negative supercoiling) and a left-handed coil is assigned a positive number (positive
supercoiling). Is there a relation between Tw and Wr? Indeed, there is. Topology tells us that the sum of Tw and Wr is
equal to Lk.
In Figure 27.19, the partly unwound circular DNA has Tw ~ 23 and Wr ~ 0, whereas the supercoiled DNA has Tw ~ 25
and Wr ~ -2. These forms can be interconverted without cleaving the DNA chain because they have the same value of
Lk; namely, 23. The partitioning of Lk (which must be an integer) between Tw and Wr (which need not be integers) is
determined by energetics. The free energy is minimized when about 70% of the change in Lk is expressed in Wr and
30% is expressed in Tw. Hence, the most stable form would be one with Tw = 24.4 and Wr = -1.4. Thus, a lowering of
Lk causes both right-handed (negative) supercoiling of the DNA axis and unwinding of the duplex. Topoisomers
differing by just 1 in Lk, and consequently by 0.7 in Wr, can be readily separated by agarose gel electrophoresis because
their hydrodynamic volumes are quite different supercoiling condenses DNA (Figure 27.20). Most naturally occurring
DNA molecules are negatively supercoiled. What is the basis for this prevalence? As already stated, negative
supercoiling arises from the unwinding or underwinding of the DNA. In essence, negative supercoiling prepares DNA
for processes requiring separation of the DNA strands, such as replication or transcription. Positive supercoiling
condenses DNA as effectively, but it makes strand separation more difficult.
Type I Topoisomerases Relax Supercoiled Structures
The interconversion of topoisomers of DNA is catalyzed by enzymes called topoisomerases which were discovered by
James Wang and Martin Gellert. These enzymes alter the linking number of DNA by catalyzing a three-step process: (1)
the cleavage of one or both strands of DNA, (2) the passage of a segment of DNA through this break, and (3) the
resealing of the DNA break. Type I topoisomerases cleave just one strand of DNA, whereas type II enzymes cleave both
strands. Both type I and type II topoisomerases play important roles in DNA replication and in transcription and
recombination.
Type I topoisomerases catalyze the relaxation of supercoiled DNA, a thermodynamically favorable process. Type II
topoisomerases utilize free energy from ATP hydrolysis to add negative supercoils to DNA. The two types of enzymes
have several common features, including the use of key tyrosine residues to form covalent links to the polynucleotide
backbone that is transiently broken.
The three-dimensional structures of several type I topoisomerases have been determined .These structures
reveal many features of the reaction mechanism. Human type I topoisomerase comprises four domains, which are
arranged around a central cavity having a diameter of 20 Å, just the correct size to accommodate a double-stranded DNA
molecule. This cavity also includes a tyrosine residue (Tyr 723), which acts as a nucleophile to cleave the DNA
backbone in the course of catalysis.
From analyses of these structures and the results of other studies, the relaxation of negatively supercoiled DNA
molecules are known to proceed in the following manner .First, the DNA molecule binds inside the cavity
of the topoisomerase. The hydroxyl group of tyrosine 723 attacks a phosphate group on one strand of the DNA backbone
to form a phosphodiester linkage between the enzyme and the DNA, cleaving the DNA and releasing a free 5 -hydroxyl
group.
With the backbone of one strand cleaved, the DNA can now rotate around the remaining strand, driven by the release of
the energy stored because of the supercoiling. The rotation of the DNA unwinds supercoils. The enzyme controls the
rotation so that the unwinding is not rapid. The free hydroxyl group of the DNA attacks the phosphotyrosine residue to
reseal the backbone and release tyrosine. The DNA is then free to dissociate from the enzyme. Thus, reversible cleavage
of one strand of the DNA allows controlled rotation to partly relax supercoiled DNA.

Type II Topoisomerases Can Introduce Negative Supercoils Through Coupling
to ATP Hydrolysis
Supercoiling requires an input of energy because a supercoiled molecule, in contrast with its relaxed counterpart, is
torsionally stressed. The introduction of an additional supercoil into a 3000-bp plasmid typically requires about 7 kcal
mol-1.
Supercoiling is catalyzed by type II topoisomerases. These elegant molecular machines couple the binding and
hydrolysis of ATP to the directed passage of one DNA double helix through another that has been temporarily cleaved.
These enzymes have several mechanistic features in common with the type I topoisomerases.
The topoisomerase II from yeast is a heart-shaped dimer with a large central cavity .This cavity has gates
at both the top and the bottom that are crucial to topoisomerase action. The reaction begins with the binding of one
double helix (hereafter referred to as the G, for gate, segment) to the enzyme .Each strand is positioned
next to a tyrosine residue, one from each monomer, capable of forming a covalent linkage with the DNA backbone. This
complex then loosely binds a second DNA double helix (hereafter referred to as the T, for transported, segment). Each
monomer of the enzyme has a domain that binds ATP; this ATP binding leads to a conformational change that strongly
favors the coming together of the two domains. As these domains come closer together, they trap the bound T segment.
This conformational change also forces the separation and cleavage of the two strands of the G segment. Each strand is
joined to the enzyme by a tyrosine-phosphodiester linkage. Unlike the type I enzymes, the type II topoisomerases hold
the DNA tightly so that it cannot rotate. The T segment then passes through the cleaved G segment and into the large
central cavity. The ligation of the G segment leads to release of the T segment through the gate at the bottom of the
enzyme. The hydrolysis of ATP and the release of ADP and orthophosphate allow the ATP-binding domains to separate,
preparing the enzyme to bind another T segment. The overall process leads to a decrease in the linking number by two.
The degree of supercoiling of DNA is thus determined by the opposing actions of two enzymes. Negative supercoils are
introduced by topoisomerase II and are relaxed by topoisomerase I. The amounts of these enzymes and their activities
are regulated to maintain an appropriate degree of negative supercoiling.
The bacterial topoisomerase II (often called DNA gyrase) is the target of several antibiotics that inhibit the
prokaryotic enzyme much more than the eukaryotic one. Novobiocin blocks the binding of ATP to gyrase.
Nalidixic acid and ciprofloxacin, in contrast, interfere with the breakage and rejoining of DNA chains. These two gyrase
inhibitors are widely used to treat urinary tract and other infections. Camptothecin, an antitumor agent, inhibits human
topoisomerase I by stabilizing the form of the enzyme covalently linked to DNA.

DNA Polymerases Require a Template and a Primer

DNA polymerases catalyze the formation of polynucleotide chains through the addition of successive nucleotides
derived from deoxynucleoside triphosphates. The polymerase reaction takes place only in the presence of an appropriate
DNA template. Each incoming nucleoside triphosphate first forms an appropriate base pair with a base in this template.
Only then does the DNA polymerase link the incoming base with the predecessor in the chain. Thus, DNA polymerases
are template-directed enzymes.
DNA polymerases add nucleotides to the 3 end of a polynucleotide chain. The polymerase catalyzes the nucleophilic
attack of the 3 -hydroxyl group terminus of the polynucleotide chain on the -phosphate group of the nucleoside
triphosphate to be added .To initiate this reaction, DNA polymerases require a primer with a free 3 -
hydroxyl group already base-paired to the template. They cannot start from scratch by adding nucleotides to a free singlestranded
DNA template. RNA polymerase, in contrast, can initiate RNA synthesis without a primer .
All DNA Polymerases Have Structural Features in Common
The three-dimensional structures of a number of DNA polymerase enzymes are known. The first such structure to be
determined was that of the so-called Klenow fragment of DNA polymerase I from E. coli. This fragment
comprises two main parts of the full enzyme, including the polymerase unit. This unit approximates the shape of a right
hand with domains that are referred to as the fingers, the thumb, and the palm. In addition to the polymerase, the Klenow
fragment includes a domain with 3 5 exonuclease activity that participates in proofreading and correcting the
polynucleotide product .
DNA polymerases are remarkably similar in overall shape, although they differ substantially in detail. At least five
structural classes have been identified; some of them are clearly homologous, whereas others are probably the
products of convergent evolution. In all cases, the finger and thumb domains wrap around DNA and hold it across the
enzyme's active site, which comprises residues primarily from the palm domain. Furthermore, all the polymerases
catalyze the same polymerase reaction, which is dependent on two metal ions.
Two Bound Metal Ions Participate in the Polymerase Reaction
Like all enzymes with nucleoside triphosphate substrates, DNA polymerases require metal ions for activity. Examination
of the structures of DNA polymerases with bound substrates and substrate analogs reveals the presence of two metal ions
in the active site. One metal ion binds both the deoxynucleoside triphosphate (dNTP) and the 3 -hydroxyl group of the
primer, whereas the other interacts only with the 3 -hydroxyl group .The two metal ions are bridged by
the carboxylate groups of two aspartate residues in the palm domain of the polymerase. These side chains hold the metal
ions in the proper position and orientation. The metal ion bound to the primer activates the 3 -hydroxyl group of the
primer, facilitating its attack on the -phosphate group of the dNTP substrate in the active site. The two metal ions
together help stabilize the negative charge that accumulates on the pentacoordinate transition state. The metal ion
initially bound to dNTP stabilizes the negative charge on the pyrophosphate product.
The Specificity of Replication Is Dictated by Hydrogen Bonding and the
Complementarity of Shape Between Bases
DNA must be replicated with high fidelity. Each base added to the growing chain should with high probability be the
Watson-Crick complement of the base in the corresponding position in the template strand. The binding of the NTP
containing the proper base is favored by the formation of a base pair, which is stabilized by specific hydrogen bonds.
The binding of a noncomplementary base is unlikely, because the interactions are unfavorable. The hydrogen bonds
linking two complementary bases make a significant contribution to the fidelity of DNA replication. However, DNA
polymerases replicate DNA more faithfully than these interactions alone can account for.
The examination of the crystal structures of various DNA polymerases indicated several additional mechanisms by
which replication fidelity is improved. First, residues of the enzyme form hydrogen bonds with the minor-groove side of
the base pair in the active site .In the minor groove, hydrogen-bond acceptors are present in the same
positions for all Watson-Crick base pairs. These interactions act as a "ruler" that measures whether a properly spaced
base pair has formed in the active site. Second, DNA polymerases close down around the incoming NTP .The binding of a nucleoside triphosphate into the active site of a DNA polymerase triggers a conformational change: the
finger domain rotates to form a tight pocket into which only a properly shaped base pair will readily fit. The mutation of
a conserved tyrosine residue at the top of the pocket results in a polymerase that is approximately 40 times as error prone
as the parent polymerase.

Many Polymerases Proofread the Newly Added Bases and Excise Errors
Many polymerases further enhance the fidelity of replication by the use of proofreading mechanisms. As already noted,
the Klenow fragment of E. coli DNA polymerase I includes an exonuclease domain that does not participate in the
polymerization reaction itself. Instead, this domain removes mismatched nucleotides from the 3 end of DNA by
hydrolysis. The exonuclease active site is 35 Å from the polymerase active site, yet it can be reached by the newly
synthesized polynucleotide chain under appropriate conditions. The proofreading mechanism relies on the increased
probability that the end of a growing strand with an incorrectly incorporated nucleotide will leave the polymerase site
and transiently move to the exonuclease site .
How does the enzyme sense whether a newly added base is correct? First, an incorrect base will not pair correctly with
the template strand. Its greater structural fluctuation, permitted by the weaker hydrogen bonding, will frequently bring
the newly synthesized strand to the exonuclease site. Second, after the addition of a new nucleotide, the DNA
translocates by one base pair into the enzyme. The newly formed base pair must be of the proper dimensions to fit into a
tight binding site and participate in hydrogen-bonding interactions in the minor groove similar to those in the
polymerization site itself .Indeed, the duplex DNA within the enzyme adopts an A-form structure,
allowing clear access to the minor groove. If an incorrect base is incorporated, the enzyme stalls, and the pause provides
additional time for the strand to migrate to the exonuclease site. There is a cost to this editing function, however: DNA
polymerase I removes approximately 1 correct nucleotide in 20 by hydrolysis. Although the removal of correct
nucleotides is slightly wasteful energetically, proofreading increases the accuracy of replication by a factor of
approximately 1000.
The Separation of DNA Strands Requires Specific Helicases and ATP
Hydrolysis
For a double-stranded DNA molecule to replicate, the two strands of the double helix must be separated from each other,
at least locally. This separation allows each strand to act as a template on which a new polynucleotide chain can be
assembled. For long double-stranded DNA molecules, the rate of spontaneous strand separation is negligibly low under
physiological conditions. Specific enzymes, termed helicases, utilize the energy of ATP hydrolysis to power strand
separation.
The detailed mechanisms of helicases are still under active investigation. However, the determination of the threedimensional
structures of several helicases has been a source of insight. For example, a bacterial helicase called PcrA
comprises four domains, hereafter referred to as domains A1, A2, B1, and B2 .Domain A1 contains a Ploop
NTPase fold, as was expected from amino acid sequence analysis. This domain participates in ATP binding and
hydrolysis. Domain B1 is homologous to domain A1 but lacks a P-loop. Domains A2 and B2 have unique structures.
From an analysis of a set of helicase crystal structures bound to nucleotide analogs and appropriate double- and singlestranded
DNA molecules, a mechanism for the action of these enzymes was proposed. Domains A1 and
B1 are capable of binding single-stranded DNA. In the absence of bound ATP, both domains are bound to DNA. The
binding of ATP triggers conformational changes in the P-loop and adjacent regions that lead to the closure of the cleft
between these two domains. To achieve this movement, domain A1 releases the DNA and slides along the DNA strand,
moving closer to domain B1. The enzyme then catalyzes the hydrolysis of ATP to form ADP and orthophosphate. On
product release, the cleft between domains A and B springs open. In this state, however, domain A1 has a tighter grip on
the DNA than does domain B1, so the DNA is pulled across domain B1 toward domain A1. The result is the
translocation of the enzyme along the DNA strand in a manner similar to the way in which an inchworm moves. In
regard to PcrA, the enzyme translocates in the 3 5 direction. When the helicase encounters a region of doublestranded
DNA, it continues to move along one strand and displaces the opposite DNA strand as it progresses.
Interactions with specific pockets on the helicase help destabilize the DNA duplex, aided by ATP-induced
conformational changes.
Helicases constitute a large and diverse class of enzymes. Some of these enzymes move in a 5 3 direction,
whereas others unwind RNA rather than DNA and participate in processes such as RNA splicing and the initiation
of mRNA translation. A comparison of the amino acid sequences of hundreds of these enzymes reveals seven regions of
striking conservation .Mapping these regions onto the PcrA structure shows that they line the ATPbinding
site and the cleft between the two domains, consistent with the notion that other helicases undergo
conformational changes analogous to those found in PcrA. However, whereas PcrA appears to function as a monomer,
other members of the helicase class function as oligomers. The hexameric structures of one important group are similar
to that of the F1 component of ATP synthase suggesting potential mechanistic similarities.

THE STRUCTURE OF DNA

Perhaps the most exciting aspect of the structure of DNA deduced by Watson and Crick was, as expressed in their words,
that the "specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic
material." A double helix separated into two single strands can be replicated because each strand serves as a template on
which its complementary strand can be assembled .Although this notion of how DNA is replicated is
absolutely correct, the doublehelical structure of DNA poses a number of challenges to replication, as does the need for
extremely faithful copying of the genetic information.
1. The two strands of the double helix have a tremendous affinity for one another, created by the cooperative effects of
the many hydrogen bonds that hold adjacent base pairs together. Thus, a mechanism is required for separating the strands
in a local region to provide access to the bases that act as templates. Specific proteins melt the double helix at specific
sites to initiate DNA replication, and other enzymes, termed helicases, use the free energy of ATP hydrolysis to move
this melted region along the double helix as replication progresses.
2. The DNA helix must be unwound to separate the two strands. The local unwinding in one region leads to stressful
overwinding in surrounding regions .Enzymes termed topoisomerases introduce supercoils that release the
strain caused by overwinding.
3. DNA replication must be highly accurate. As noted in Chapter 5, the free energies associated with base pairing within
the double helix suggest that approximately 1 in 104 bases incorporated will be incorrect. Yet, DNA replication has an
error rate estimated to be 1 per 1010 nucleotides. As we shall see, additional mechanisms allow proofreading of the
newly formed double helix.
4. DNA replication must be very rapid, given the sizes of the genomes and the rates of cell division. The E. coli genome
contains 4.8 million base pairs and is copied in less than 40 minutes. Thus, 2000 bases are incorporated per second. We
shall examine some of the properties of the macromolecular machines that replicate DNA with such high accuracy and
speed.
5. The enzymes that copy DNA polymerize nucleotides in the 5 3 direction. The two polynucleotide strands of DNA
run in opposite directions, yet both strands appear to grow in the same direction .Further analysis reveals
that one strand is synthesized in a continuous fashion, whereas the opposite strand is synthesized in fragments in a
discontinuous fashion. The synthesis of each fragment must be initiated in an independent manner, and then the
fragments must be linked together. The DNA replication apparatus includes enzymes for these priming and ligation
reactions.
6. The replication machinery alone cannot replicate the ends of linear DNA molecules, so a mechanism is required to
prevent the loss of sequence information with each replication. Specialized structures called telomeres are added by
another enzyme to maintain the information content at chromosome ends.
7. Most components of the DNA replication machinery serve to preserve the integrity of a DNA sequence to the
maximum possible extent, yet a variety of biological processes require DNA formed by the exchange of material
between two parent molecules. These processes range from the development of diverse antibody sequences in the
immune system to the integration of viral genomes into host DNA. Specific enzymes, termed
recombinases, facilitate these rearrangements.
8. After replication, ultraviolet light and a range of chemical species can damage DNA in a variety of ways. All
organisms have enzymes for detecting and repairing harmful DNA modifications. Agents that introduce chemical lesions
into DNA are key factors in the development of cancer, as are defects in the repair systems that correct these lesions.
We begin with a review of the structural properties of the DNA double helix.
III. Synthesizing the Molecules of Life 27. DNA Replication, Recombination, and Repair
III. Synthesizing the Molecules of Life 27. DNA Replication, Recombination, and Repair
DNA Can Assume a Variety of Structural Forms
The double-helical structure of DNA deduced by Watson and Crick immediately suggested how genetic information is
stored and replicated. As was discussed earlier the essential features of their model are:
1. Two polynucleotide chains running in opposite directions coil around a common axis to form a right-handed double
helix.
2. Purine and pyrimidine bases are on the inside of the helix, whereas phosphate and deoxyribose units are on the outside.
3. Adenine (A) is paired with thymine (T), and guanine (G) with cytosine (C). An A-T base pair is held together by two
hydrogen bonds, and that of a G-C base pair by three such bonds.

A-DNA Is a Double Helix with Different Characteristics from Those of the More
Common B-DNA
Watson and Crick based their model (known as the B-DNA helix) on x-ray diffraction patterns of DNA fibers, which
provided information about properties of the double helix that are averaged over its constituent residues. The results of xray
diffraction studies of dehydrated DNA fibers revealed a different form called A-DNA, which appears when the
relative humidity is reduced to less than about 75%. A-DNA, like B-DNA, is a right-handed double helix made up of
antiparallel strands held together by Watson-Crick base-pairing. The A helix is wider and shorter than the B helix, and its
base pairs are tilted rather than perpendicular to the helix axis (Figure 27.4).
Many of the structural differences between B-DNA and A-DNA arise from different puckerings of their ribose units
In A-DNA, C-3 lies out of the plane (a conformation referred to as C-3 -endo) formed by the other four
atoms of the furanose ring; in B-DNA, C-2 lies out of the plane (a conformation called C-2 -endo). The C-3 -endo
puckering in A-DNA leads to a 19-degree tilting of the base pairs away from the normal to the helix. The phosphates and
other groups in the A helix bind fewer H2O molecules than do those in B-DNA. Hence, dehydration favors the A form.
The A helix is not confined to dehydrated DNA. Double-stranded regions of RNA and at least some RNA-DNA hybrids
adopt a double-helical form very similar to that of A-DNA. The position of the 2 -hydroxyl group of ribose prevents
RNA from forming a classic Watson-Crick B helix because of steric hindrance the 2 -oxygen atom would
come too close to three atoms of the adjoining phosphate group and one atom in the next base. In an A-type helix, in
contrast, the 2 -oxygen projects outward, away from other atoms.
The Major and Minor Grooves Are Lined by Sequence-Specific Hydrogen-
Bonding Groups
Double-helical nucleic acid molecules contain two grooves, called the major groove and the minor groove. These
grooves arise because the glycosidic bonds of a base pair are not diametrically opposite each other The
minor groove contains the pyrimidine O-2 and the purine N-3 of the base pair, and the major groove is on the opposite
side of the pair. The methyl group of thymine also lies in the major groove. In B-DNA, the major groove is wider (12
versus 6 Å) and deeper (8.5 versus 7.5 Å) than the minor groove .Each groove is lined by potential hydrogen-bond donor and acceptor atoms that enable specific interactions with proteins
. In the minor groove, N-3 of adenine or guanine and O-2 of thymine or cytosine can serve as hydrogen
acceptors, and the amino group attached to C-2 of guanine can be a hydrogen donor. In the major groove, N-7 of guanine
or adenine is a potential acceptor, as are O-4 of thymine and O-6 of guanine. The amino groups attached to C-6 of
adenine and C-4 of cytosine can serve as hydrogen donors. Note that the major groove displays more features that
distinguish one base pair from another than does the minor groove. The larger size of the major groove in B-DNA makes
it more accessible for interactions with proteins that recognize specific DNA sequences.
The Results of Studies of Single Crystals of DNA Revealed Local Variations in
DNA Structure
X-ray analyses of single crystals of DNA oligomers had to await the development of techniques for synthesizing large
amounts of DNA fragments with defined base sequences. X-ray analyses of single crystals of DNA at atomic resolution
revealed that DNA exhibits much more structural variability and diversity than formerly envisaged.
The x-ray analysis of a crystallized DNA dodecamer by Richard Dickerson and his coworkers revealed that its overall
structure is very much like a B-form Watson-Crick double helix. However, the dodecamer differs from the Watson-Crick
model in not being uniform; there are rather large local deviations from the average structure. The Watson-Crick model
has 10 residues per complete turn, and so a residue is related to the next along a chain by a rotation of 36 degrees. In
Dickerson's dodecamer, the rotation angles range from 28 degrees (less tightly wound) to 42 degrees (more tightly
wound). Furthermore, the two bases of many base pairs are not perfectly coplanar .Rather, they are
arranged like the blades of a propeller. This deviation from the idealized structure, called propeller twisting, enhances
the stacking of bases along a strand. These and other local variations of the double helix depend on base sequence. A
protein searching for a specific target sequence in DNA may sense its presence through its effect on the precise shape of
the double helix.
Z-DNA Is a Left-Handed Double Helix in Which Backbone Phosphates Zigzag
Alexander Rich and his associates discovered a third type of DNA helix when they solved the structure of dCGCGCG.
They found that this hexanucleotide forms a duplex of antiparallel strands held together by Watson-Crick base-pairing,
as expected. What was surprising, however, was that this double helix was left-handed, in contrast with the right-handed
screw sense of the A and B helices. Furthermore, the phosphates in the backbone zigzagged; hence, they called this new
form Z-DNA .The Z-DNA form is adopted by short oligonucleotides that have sequences of alternating pyrimidines and purines. High
salt concentrations are required to minimize electrostatic repulsion between the backbone phosphates, which are closer to
each other than in A- and B-DNA. Under physiological conditions, most DNA is in the B form. Although the biological
role of Z-DNA is still under investigation, its existence graphically shows that DNA is a flexible, dynamic molecule.

STREPTOCOCCUS

Gram positive cocci arranged in chains or pairs.
They cause pyogenic infections with a characteristic tendency to spread unlike staph(which typically localize)
Billroth called them streptococci (Strepto meaning twisted or coiled)
They cause Non Suppurative lesions, acute rheumatic fever & glomerulonephritis.
Streptococci are first divided into obligate anaerobes & facultative anaerobes.
Streptococci are classified on basis of hemolytic properties; Alpha , Beta , Gamma.
Most pathogenic strepto fall into group Beta & called as hemolytic strepto cocci.
Alpha strepto cocci are generally commensals in throat and produce opportunistic infections called as viridance group(green).
Gamma strepto cocci include fecal strepto cocci.

MORPHOLOGY
Gram positive cocci in chains , 0.5 – 1 um in diameter.
They are non motile and non sporing.
Group C strains have some capsules.

CULTURE
Aerobic and facultative anaerobe.
Grows best at 37 degrees C (range – 22 to 42 degree C)
They have exact nutritive requirements, growth occurring in media with blood ,serum or sugars.
Colonies are small (0.5 to 1 mm) circular ,semi transparent & low convex with a wide zone of beta hemolysis.
Hemolysis is promoted by 10% CO2.
Virulent strains form “matt” colonies,
Avirulent strains form glossy colonies.

RESISTANCE
Delicate organisms and inactivated by heat at 56 degree C for 30 minutes.
They survive in dust for several weeks if protected from sunlight.
It is rapidly inactivated by antiseptics.
Its more resistant to crystal violet than many other bacteria.
They do not develop resistance to drugs. (unlike staph)
They are sensitive to bacitracin.

ANTIGENIC STUCTURE
1) Capsular Hyaluronic Acid
2)Group Specific Polysaccharide Antigen
3)Type Specific Antigen
M,T&R proteins

TOXINS AND OTHER VIRULENCE FACTORS
Hemolysins
There are two types of hemolysins: O and S
Streptolysin O:
Lyses red cells ,cytotoxic for neutrophils platelets and cardiac tissue.
It is antigenic and antistreptolysin O appears in sera post streptococal infection.

Estimation (ASO titre) standard serological procedure for the retrospective diagnosis.
ASO titre > 200 units suggest either recent or recurrent streptococcal infection.
Streptolysin S:
Oxygen stable.
Responsible for haemolysis in blood agar.
It is a protein but not antigenic.
Pyrogenic exotoxin (erythrogenic, dick, scarlatinal toxin)
Three types of streptococcal pyrogenic exotoxin (S P E) A,B & C
SPE ‘s are SUPERANTIGENS (induce massive release of cytokines causing fever, shock & tissue damage)
Streptokinase(Fibrinolysin)
It breaks down fibrin barrier around lesions and spreads the infection.
Streptokinase IV given as RX in early myocardial infarction and other thromboembolic disoders.

Deoxyribonucleases(stretodornase,DNAase)
Stretodornase liquefies the thick pus.
This property apply therapeutically in liquefying thick exudates as in empyema.
There are four types of DNAases A,B,C &D.
Nicotinamide adenine dinucleotidase (NADase, formerly diphosphopyridine nucleotidase, DPNase)
It is leucotoxic.
It is antigenic.
Hyaluronidase
Breaks down hyaluronic acid of tissues and spread the infection along intercellular spaces.
It is semingly self destructive process.
It is antigenic.
Serum opacity factor (SOP
PATHOGENICITY
Streptococcus pyogenes is intrinsically much more dangerous organism than staph aureus and has much greater tendency to spread in the tissues.
It is more likely to give septicaemia.
Carriers(5% )of general population carry S pyogenes in resp tract , mouth & skin.
Carrier rate is higher in children between 1 to 15 years of age.
Carriers and patients with acute infections are the sources of infection.

Treatment of Syphilis During Pregnancy

Penicillin is the drug of choice for treating all stages of syphilis. Parenteral rather than oral treatment has been the route of choice as the therapy is supervised and bioavailability is guaranteed. Most women treated during pregnancy will deliver before their serological response to treatment can be assessed definitively. Neonates born to such women should be evaluated for congenital syphilis. The UK national guidelines for the treatment of early syphilis during pregnancy are described as follows:
First-line therapy: intramuscular (i.m.) procaine penicillin 750 mg daily for 10 days. If it is not possible to give daily procaine penicillin on the weekend, then either long-acting procaine penicillin in aluminium stearate, 2 million units (MU) or long-acting benethamine penicillin 1.2 MU should be given IM on the Friday.
Patients with penicillin allergy: erythromycin 500 mg four times a day should be given for 14 days. Alternatively, azithromycin 500 mg should be given daily for 10 days. In addition to this, examination, tests, and treatment of all babies at birth should be carried out. Desensitization to penicillin may be considered, followed by the first-line treatment. Mothers treated with erythromycin or azithromycin may be considered for retreatment with doxycycline after delivery and when breast-feeding is stopped.
Patients suspected of non-compliance: benzathine penicillin 2.4 MU i.m. on Days 1 and 8.
Penicillin Reactions
Approximately 5–10% of pregnant women with syphilis report a history of penicillin allergy. The Jarisch–Herxheimer reaction is an acute response that may occur after treatment for acquired early syphilis. It occurs in up to 45% of pregnant women and consists of fever, chills, myalgia, headache, hypotension, tachycardia, and transient accentuation of the cutaneous lesions.[6] It typically begins within several hours of treatment and resolves within 24–36 h. The release of T. pallidum lipoprotein, which possesses inflammatory activity from dead or dying organisms, is implicated as a likely inducer of this phenomenon. In pregnant women, the Jarisch–Herxheimer reaction can cause uterine contractions and precipitate labour. This is possibly mediated secondarily by prostaglandins as the concentrations are increased during reactions.[6,10]
Syphilis and HIV
Syphilis commonly co-exists in patients with HIV (prevalence is 14–36%). All HIV-infected patients under regular follow-up should have syphilis serology documented at baseline and subsequently 12 monthly thereafter. HIV-infected patients with early syphilis have an increased risk of neurological involvement. Features of syphilis in HIV include: generalized lymphadenopathy; splenomegaly; hepatitis; skin rashes, alopecia or both; oral manifestations; cognitive impairment; meningitis; cranial nerve palsies; myopathies; and uveitis.
Anaesthetic Considerations
There is little specific advice available on the anaesthetic management of patients with syphilis. Universal precautions should be considered at all times when anaesthetizing patients with syphilis. Accidental transmission of syphilis involves direct contact through a small skin abrasion. It has been reported under the following circumstances: doctors and nurses who have examined a syphilitic lesion without wearing gloves; laboratory workers by needle stick injury when inoculating treponemes into rabbits, or during isolation or purification procedures; and patients being transfused with blood from a donor suffering from early syphilis.
Infection by blood transfusion is rare in the UK because screening tests are routinely performed for evidence of donor infection with syphilis. After storage in blood for more than 4 days at 4oC, spirochetes are non-viable. The risk of accidental infection by infected blood is highest when fresh heparinized blood is used. Such blood is used for exchange transfusion in neonates. Cutaneous lesions of the breast and nipples carry a risk of transmission through breast feeding. After needle-stick injury, the risk of transmission is very low. Antibiotics are not routinely recommended for needle-stick injuries; however, each wound should be assessed individually by the relevant healthcare professionals.
There is no additional risk with general anaesthesia. There is a single report of a 73-year-old woman with late congenital pharyngo-laryngeal syphilis, who presented with a potentially difficult intubation during the induction of general anaesthesia.[11] Syphilis poses no specific problems for regional blockade. The three main manifestations of late syphilis (neuro-, cardiovascular, and gummatous syphilis) can have a wide range of presentation. It is prudent to assess and document all existing signs and symptoms (including neurological examination) in the anaesthetic record. There is no evidence to suggest that regional blockade can affect the extent or likelihood of neurosyphilis. The lesion in tabes dorsalis is concentrated on the dorsal spinal roots and dorsal columns of the spinal cord, most often at the lumbosacral and the lower thoracic region. There have been reports that spinal anaesthesia induces severe lightning pain in the lower limbs of patients with phantom limb pain, tabes dorsalis, or causalgia. The exact mechanism of this phenomenon is controversial. Some hypothesize that complete loss of sensory input after spinal anaesthesia may decrease the level of inhibition and increase the self-sustained neural activity.
Options for delivery include elective Caesarean section because it is associated with less vertical transmission. When considering postoperative analgesia, those techniques that do not expose staff to needle-stick injury should be favoured.

Laboratory Diagnosis of Syphilis

Laboratory Diagnosis of Syphilis
Diagnosis of syphilis is based on microscopy and serology. At the first antenatal visit, all women in UK are screened for sexually transmitted diseases including syphilis and HIV. The serological tests are repeated at three monthly intervals in cases of anogenital ulceration if the initial tests are negative. All infants born to seropositive mothers should be examined at birth and at monthly intervals for 3 months until it is confirmed that serological tests are and remain negative.
Microscopy
Microscopic demonstration of T. pallidum from the lesions or infected lymph nodes in early syphilis depends on the following three tests:
Dark-field microscopy: if a lesion such as chancre is present, dark-field microscopy should be attempted to visualize the characteristic motile spirochetes in the exudates collected from the lesion. The sensitivity rate[5] is up to 97%, so failure to find the organism does not exclude a diagnosis of syphilis. (For an explanation of sensitivity and specificity, please see Lalkhen and McCluskey.[8])
Direct fluorescent antibody (DFA) test: this uses the indirect fluorescent technique with killed T. pallidum as antigen. The organisms are fixed on a slide to which serum is added. The antibody in the serum unites with treponemes and is made visible with fluorescent stain.[1]
Polymerase chain reaction (PCR) test: it may be useful for the detection of primary syphilis with sensitivity up to 98.6%.
Serological Tests
Non-treponemal Tests. These tests detect the cross-reaction of antibody to syphilis with cardiolipin. The result is reported as reactive or non-reactive; a reactive test is accompanied by a quantitative titre and should be confirmed with a treponemal test. False positive non-treponemal tests may occur in patients who are pregnant, i.v. drug users, those with systemic inflammatory diseases such as systemic lupus erythematosus, or after a recent viral infection.[3]
VDRL (venereal disease research laboratory) test: this is simple and inexpensive and is the preferred test worldwide.
RPR (rapid plasma reagin) test: this is used for screening purposes and is the least technically demanding test as no microscope is needed. It uses carbon-containing cardiolipin antigen and requires a minimal quantity of blood.
Treponemal Tests. These tests specifically detect antibodies against T. pallidum and are positive for life in the vast majority of infected patients regardless of stage or treatment history.[3]
TPHA (T. pallidum haemagglutination assay) or TPPA (T. pallidum particle agglutination assay): these are very valuable and simple tests using an indirect haemagglutination method with red cells or by gelatine particles. Together with VDRL, it is probably the best combination for routine use. False positive reactions occur in up to 2%.[1]
EIA (enzyme immuno assay): treponemal enzyme immunoassay is the screening test of choice and can detect IgG and IgM antibodies as it is positive in earlier stages of syphilis. A positive test is then confirmed with the TPHA/TPPA or VDRL/RPR tests.
FTA-ABS (fluorescent treponemal antibody absorption) assay: this uses the indirect fluorescent technique with killed T. pallidum as an antigen. The organisms are fixed on a slide to which serum is added. The antibody in the serum unites with treponemes. The test has been made more specific by absorbing the group antibodies. This is the most sensitive and specific test available. It becomes positive earlier during the initial stage of primary syphilis. However, it is not suitable for assessing the activity, as the positive test persists long after successful treatment.[1]
Neurological involvement[9] is confirmed by a positive VDRL, raised cell count (>5/mm2), and raised protein (40 mg dl–1) in the CSF obtained by lumbar puncture. Chest X-ray, electrocardiography, echocardiography, cardiac catheterization, and biopsy of gumma can reveal involvement of other systems.

The Microbiological Disease Syphilis in Pregnancy-Clasification

Introduction
For several decades, syphilis has been out of sight, mind, and memory, but the incidence in the Western world is now on the rise again and it could once more become a major health concern. This change has followed the rapidly rising number of human immunodeficiency virus (HIV) positive individuals worldwide, together with the advent of health tourists, economic migrants, asylum seekers, and the easy availability of low-cost travel.
Just as syphilis has all but disappeared as an entity in the working memory of most anaesthetists, it has suddenly re-emerged as a co-existing condition in women presenting for Caesarean section.
Incidence of Syphilis
The 1999 WHO estimates suggest an annual rate for syphilis of ~12 million active infections. The risk of contracting syphilis through a sexual contact with a person with primary or secondary syphilis is 30–50%. More than 80% of women with syphilis are in reproductive age; therefore, there is a serious risk of vertical transmission to the fetus.[6] Worldwide, a million pregnancies are adversely affected each year by syphilis because of maternal infection. About 270 000 babies are born with congenital syphilis, 460 000 pregnancies end in abortion or perinatal death, and 270 000 babies are born prematurely or with low birth weight.[7]

Aetiology[1]
Treponema pallidum is the causative organism for syphilis. It is a delicate, motile spirochete bacterium. Humans are its only natural source. Syphilis is usually transmitted by sexual contact through exposure to mucocutaneous syphilitic lesions that contain infectious spirochetes. The infecting organism in body fluid gains access through microscopic abrasions in skin or mucosal surfaces, and begins to replicate locally. After inoculation, the incubation period is around 3 weeks (10–90 days), at the end of which a primary sore develops at the site of infection, usually the genitalia.

Classification
Syphilis is classified[2] as congenital or acquired. There are four stages of syphilis: primary, secondary, latent, and late (tertiary).

Primary Syphilis
The first development is a chancre at the site of inoculation, classically in the anogenital region which is a painless, solitary, round indurated ulcer with a bright red margin.[1] Chancres appear on average about 3 weeks after sexual contact and heal in 3–6 weeks. However, with a small inoculum, this incubation period may be as long as 90 days. One of the common sites for lesions is the cervix; therefore, the clinical manifestations of primary syphilis may go unnoticed by the patient and her partner.[3]
Secondary Syphilis
Untreated patients will progress to secondary syphilis after the signs for primary syphilis resolve (within 4–10 weeks[3]). The lesions are numerous, variable, and affect many systems. A symmetrically distributed, maculopapular, non-irritating rash is found on the palms and the soles with painless lymphadenopathy. The highly infectious condyloma lata are found on warm and moist areas such as genitalia, perianal region, perineum, and axillae. Both meningism and headache can occur, especially at night. Their cause can be confirmed by the presence of an elevated cell count and elevated proteins in cerebrospinal fluid. Less common accompaniments to secondary syphilis include alopecia, laryngitis, mild hepatitis, nephrotic syndrome, bone pain, and uveitis.
Latent Syphilis
The natural history of untreated secondary syphilis is marked by spontaneous resolution after a period of 3–12 weeks, leaving the patient entirely free of symptoms. This naturally attained asymptomatic state is called latency.[4] The latency is arbitrarily subdivided into early (<2 yr from the onset of the infection) and late (>2 yr) stages. During this time, the patient remains serologically positive for syphilis. Approximately 60% of patients remain latent for the rest of their lives. In the early latent stage, 25% will relapse with a secondary syphilitic manifestation, whereas the likelihood of such relapses in the late latent stage is small.[1]
Late Syphilis (Tertiary Syphilis)
Tertiary syphilis develops in 30–40% of untreated patients. The three main manifestations of late syphilis are cardiovascular, gummatous, and neurosyphilis. Cardiovascular syphilis usually occurs 15–30 yr after primary syphilis and may occur in any large vessel. It is characterized, by an aortitis, aortic incompetence, coronary ostial stenosis (presenting as angina), and aortic medial necrosis causing aortic aneurysm. Gummatous syphilis is granulomatous locally destructive lesions that usually occur 3–12 yr after inoculation. They can occur in almost any tissue. Neurosyphilis presents with a variety of syndromes including general paresis, tabes dorsalis, syphilitic meningitis, and meningovascular syphilis. The incubation period is 5–12 yr.[5]
Syphilis in Pregnancy
Antenatal syphilis poses a significant threat to the pregnancy and fetus. T. pallidum readily crosses the placenta, resulting in fetal infection. Vertical transmission can occur at any time during pregnancy and at any stage of syphilis.[6] Risk of transmission correlates with the extent of spirochetal presence in the circulation. Vertical transmission of syphilis is more common in primary (50%) and secondary syphilis (50%), compared with early latent (40%), late latent (10%), and tertiary syphilis (10%). Seventy to one hundred per cent of infants born to untreated infected mothers are infected. Pregnancies complicated by syphilis may result in intrauterine growth restriction, non-immune hydrops fetalis, stillbirth, preterm delivery, and spontaneous abortion in up to 50% of pregnancies. Women who had documented treatment for syphilis in the past do not need treatment during current or subsequent pregnancies.
Congenital Syphilis
In spite of a downward trend in the incidence of syphilis, congenital syphilis, an infection passed from mother to child through the placenta during fetal development or birth, remains a great concern. An infected woman’s potential to infect her fetus remains for many years, although the risk of infecting a fetus declines gradually during the course of untreated illness. After 8 yr, there is little risk, even in the untreated mother. Nearly half of all children infected with syphilis during gestation die shortly before or after birth.
Infants who survive develop early-stage and late-stage symptoms of syphilis, if not treated. Early-stage symptoms include irritability, failure to thrive, non-specific fever, a rash and condyloma lata on the borders of the mouth, anus, and genitalia. Some of these lesions may resemble the wart-like lesions of adult syphilis. A small percentage of infants have a watery nasal discharge (sniffles) and a saddle nose deformity resulting from destruction of the cartilage of the nose. Bone lesions are common, especially in the upper arm (humerus). Later signs appear as tooth abnormalities (Hutchinson teeth), bone changes (sabre shins), neurological involvement, blindness, and deafness.

DNA-Transcription regulation

HIPPI (HIP-1 protein interactor), also known as ESRRBL1 (estrogen-related receptor beta like 1), a homolog of Chlamydomonas intraflagellar transport 57 (IFT57), does not have any known domain except a ‘pseudo death effector domain’ (pDED) and a myosin like domain (MLD). Interaction of HIPPI with HIP-1 is through the pDED, specifically through 409 K, present in the putative helix5 of HIPPI-pDED, although other regions might have influence on such interactions . HIPPI-HIP-1 heterodimer recruits procaspase-8 and activates the initiator caspase and its downstream apoptotic cascades . It has been shown earlier that the strength of interaction of HIP-1 with Huntingtin (HTT) protein, whose mutation causes Huntington’s disease (HD), is inversely correlated with the number of glutamines (Q) at the N-terminal region of HTT . It is proposed that weaker interaction of HIP-1 with mutated HTT in HD might increase the freely available pool of HIP-1 and might, in turn, enhance the propensity of hetero-dimerization of HIP-1 with HIPPI. The elevated pool of HIPPI-HIP-1 complex may then recruit procaspase-8 and lead to increased cell death as observed in HD .

In addition to increased apoptosis by the activation of different caspases, truncation of Bid, release of AIF from the mitochondria, endogenous expressions of caspase-1, -3, -7 and -8 are also increased in GFP-Hippi expressing Neuro2A and HeLa cells, whereas mitochondrial genes ND1, ND4 and anti-apoptotic gene Bcl-2 are down regulated (2). We have subsequently shown that HIPPI can directly interact both in vitro and in vivo with a 60 bp sequence (–151 to –92) upstream of the caspase-1 gene. HIPPI, especially its C-terminal pDED, interacts with the specific sequence motif AAAGACATG (–101 to –93) present at the promoter sequence of caspase-1 . Similar motifs are also present at the putative promoter sequences of caspase-8 and caspase-10. HIPPI interacts with these promoters and increases the expression of these genes . This result indicates that HIPPI, without having any known DNA-binding domain, interacts with DNA and regulates transcription. Specific amino acid(s) that interact with the DNA sequence still remains unknown. Besides, the question of nuclear translocation of cytoplasmic HIPPI for transcription regulation, without having classical nuclear localization signal (NLS) is yet to be resolved.

HIP-1, the molecular partner of HIPPI interacts with membranes, traffics endocytic vesicles and translocates into the nucleus using its own NLS at the C-terminus and has been implicated in cancer . HIP-1 interacts directly with androgen receptor (AR), accumulates in the nucleus upon androgen stimulation and recruits to DNA elements regulated by AR. AR also translocates to the nucleus in response to androgen and the process is facilitated by HIP-1 . HIP-1 thus regulates the transcription of AR responsive genes through its interaction with AR. Given that HIP-1 can act as the nuclear transporter for AR and regulate the expressions of its target genes, we tested the hypothesis that HIPPI might also be translocated to the nucleus assisted by HIP-1 and could regulate the expression of caspase-1 gene.


Antibodies and other reagents
RNase A, BSA, Geniticin, Hygromycin, DAPI, Hoechst, nuclei isolation kit, anti-Beta-actin (A2228, clone AC-74, Lot number: 107K4791) antibody and Protein G were obtained from Sigma Chemicals (MO, USA). Assay kit for detection of caspase-8 activation was obtained from Alexis Biochemicals, Switzerland. The anti-mouse and anti-rabbit secondary antibodies conjugated with horseradish peroxidase, TRITC and FITC conjugated antibodies were purchased from Bangalore Genei, India; anti-GFP antibody was purchased from BD Biosciences, USA (632375, Lot number: B7040316); anti-histone 2B (H2B) antibody (IMG-359 Lot number: 073101A) and anti-caspase-1 antibody (IMG-804-4, Lot number: AB093004A) were from Imgenex, USA; anti-HIP-1 was purchased from Novus Biologicals (NB300-204, 1B11, Lot number: A); and anti-HIPPI antibody (ab5205-100, Lot number: 63362) and anti-LaminB antibody (ab16048-25, Lot Number 393854) were purchased from Abcam, USA. Immobilon-P Transfer membrane was from Millipore, USA, Chemilumiscence kit from Pierce, USA, Taq polymerase from Bioline, USA, and restriction enzymes (BamHI, SalI, SmaI, XhoI and HindIII) were from Promega, USA. Protease inhibitor cocktail was purchased from Roche, USA. Other molecular biology grade fine chemicals were procured locally.

Methods for modeling and prediction for DNA-binding property
Comparative structure-based modeling was done using MODELLER 8v1 with a multiple structural alignment template, generated by CEMC using 3D co-ordinates of all DED containing proteins (CATH homologous super family 1.10.533.10 [EC] ) from PDB . Out of twenty energy-minimized models, one with the minimum objective function was chosen for generating accessible molecular surface with electrostatic potential map using GRASP . Prediction of DNA-binding property was made using PreDs, by submitting the coordinates of modeled pDED of HIPPI and its mutant protein via the online tool using default parameters (http://pre-s.protein.osaka-u.ac.jp/~preds/). The tool makes prediction of dsDNA-binding site on protein surfaces. The prediction for the query protein is made based on the value of the prediction score, Pscore, which is a vectorial property of the surface and an indicator of the ratio of the predicted area (Parea) to the whole area on the protein surface. The protein with a higher Pscore than 0.12 is considered as a dsDNA-binding protein.

Construction of clones
Constructions of GFP-Hippi and GFP-pDED (coding for 335 to 429 amino acids of HIPPI) have been described earlier . The HIP-1 clone in pcDNA3 (pcDNA3 Hip-1) was kindly provided by Prof. T.S Ross, University of Michigan Medical School, USA. The mutant HIP-1 containing a glutamic acid (E) at the position 58 and 1005 (designated as Hip-1 58E and Hip-1 1005E) clones in pcDNA3 were kindly provided by Dr. Ian Mills, CRUK Uro-Oncology Research Group, Cambridge, CB2 0RE, UK. Full length Hip-1, N terminus Hip-1 (Hip-1N, 1–258aa) and pDED of Hip-1 (Hip-1P, 410–491aa) were sub-cloned in DsRed C1 vector. The details of the primer sequence, PCR condition and restriction enzymes used .




Site directed mutagenesis
A mutation [replacing the Arginin (R) residue at 393 position of HIPPI (59th position of pDED of HIPPI), by Aspartic acid (E) residue (AGA to GGA)] was introduced in GFP-pDED of HIPPI by PCR directed site-specific mutagenesis with GFP-pDED as a template. The recombinant GFP-pDED was subjected to PCR-directed mutagenesis using mutagenic oligonucleotide primers containing mismatch bases. The mutagenic PCR involved the generation of two PCR products that overlap in sequence containing the same mutation introduced as part of the PCR primers. A subsequent re-amplification of these fragments with cloning primers as described earlier resulted in the enrichment of the full-length pDED-HIPPI.

MutR59EF: 5'-GACATTgaAATTGGCATTGTGG-3'

MutR59ER: 5'-GCCAATTtcAATGTCCATCT-3'

The mutation thus introduced was confirmed by sequencing. The plasmid harboring the mutated gene was designated as GFP-mpDED.

Cell culture and transfection
HeLa and Neuro2A cells were routinely grown in MEM (HIMEDIA, India) while K562 cells were grown in RPMI (HIMEDIA, India) supplemented with 10% fetal bovine serum (Biowest, USA) at 37°C in 5% CO2 atmosphere under humidified condition.

Transfection of cells was performed using Lipofectamine 2000 (Invitrogen, USA). Unless otherwise mentioned, for single transfection experiment 2 µg (60 mm plate) or 5 µg (100 mm plate) of DNA constructs as well as 7 or 15 µl of Lipofectamine 2000 respectively were used. For co-transfection, equal amount of DNA constructs (5 µg each for 100 mm plate) were used. After 24 h, transiently transfected cells were checked for transfection efficiency by monitoring GFP or DsRed expression under fluorescence microscope and were used for experiments. Transfection efficiency varied from 70 to 90%.

To generate cell lines that stably express wild type and mutant HIP-1, Neuro2A cells were grown in 100 mm plates to ~30% confluency. Cells were then transfected using 5 µl of Lipofectamine 2000 and 2 µg of the required plasmid [pcDNA3Hip-1, Hip-1 1005E, Hip-1 58E]. Transfected cells were then selected using G418 (final concentration 0.4 mg/ml). After 10–15 days, clones were pooled and grown in the presence of G418. Before the experiment selection was withdrawn and used for the studies.

Knockdown of HIP-1 in HeLa and Neuro2A cells by siRNA
DNA sequences 779-ACCGCTTCATGGAGCAGTTTA-799 and 1394-ACAGCGATATAGCAAGCTAAA-1415 of human Hip-1 (gi|38045918|ref|NM_005338.4|) were designed for the siRNAs using the online software from GenScript (https://www.genscript.com/ssl-bin/app/rnai). The scrambled sequence (5'-TAGTCGCATACGGAACATTCG-3') for the first siRNA was also designed using GenScript sequence scrambler tool. The complete sequence which was inserted into the expression vector pRNATin-H1.2/Hygro was 5'-TAAACTGCTCCATGAAGCGGTTTGATATCCGACCGCTTCATGGAGCAGTTTATTTTTTCCAA-3' (designated Hip1Si) with termination signal and appropriate restriction site linkers (BamH1 and HindIII, not shown) and an insert for loop formation (underlined). Similarly for a second siRNA to target the human Hip-1 the complete sequence 5'-TTTAGCTTGCTATATCGCTGTTTGATATCCGACAGCGATATAGCAAGCTAAATTTTTTCCAA-3' (designated as Hip1Si1) was cloned as above. The entire sequence for the scramble siRNA was 5'-CGAATGTTCCGTATGCGACTATTGATATCCGTAGTCGCATACGGAACATTCGTTTTTTCCAA-3' (designated as Hip1Scr). The cloned fragments were sequenced and confirmed. The cloned DNA fragments (Hip1Si and Hip1Si1) were purchased from GeneSript, USA and the Hip1Scr was cloned in our laboratory using the restriction enzymes BamH1 and HindIII. The purchased clones were further checked with the recommended restriction enzymes (BamH1 and HindIII) digestion and used for our experiments. Mouse HIP-1 (gi|22122460|NM_146001) sequences contain a single mismatch (410-ACCGCTTCATGGAGCAGTTCA-430 of mouse HIP-1) compared with that of in human (779-ACCGCTTCATGGAGCAGTTTA-799).

HIP-1 siRNA clones and the scramble siRNA were transfected in HeLa cells using Lipofectamin2000 (Invitrogen, USA) using protocol provided by the manufacturer. Transfected cells were selected for Hygromycin resistance. Colonies grown in presence of Hygromycin were pooled and grown to sufficient numbers for protein isolation and other experiments. Protein was isolated and western blot analysis was carried out using anti HIP-1 antibody. Similarly for Neuro2A cells, Hip1Si was transfected to knock down HIP-1. Similar down regulation of HIP-1 was observed in spite of a single mismatch in siRNA sequence. In parallel, HeLa cells were also transfected with the empty vector (pRNATin-H1.2/Hygro, without the insert) and selected for Hygromycin resistance and used to check the expression of HIP-1. No change in the expression of HIP-1 was observed with cells expressing the empty vector. In all our experiments, we have used HeLa cells or Neuro2A cells as the control, while studying the effect of HIP-1 (in knocked down cells).

Detection of apoptosis by nuclear fragmentation and caspase-8 activation
Nuclear fragmentation and caspase-8 activation were detected using the methods described earlier (2). In brief, cells were grown on cover slips, washed thrice with PBS and fixed with 1:1 mixture of methanol and acetone (1 h at 4°C). Cells were then stained with 1 mM Hoechst in phosphate buffer saline (PBS) in the dark at room temperature for 5 min and observed under a fluorescence microscope (Olympus BX60 with appropriate attachment, Japan). Cells with intact nuclear morphology (normal) and fragmented nuclei (apoptotic cells) were determined and the percentage of cells with apoptotic nuclei was calculated. About 200–500 cells were counted for each experiment. Activation of caspase-8 was determined according to the protocols provided by the manufacturers of the kit. Exponentially growing 2 million HeLa cells expressing GFP–wpDED and GFP-mpDED were collected and processed as mentioned earlier . For the caspase-8 detection, the fluorescence of liberated AFC was measured at its emission maxima ({lambda}max 505 nm) with the excitation at the 400 nm.

Confocal microscopy
Cells were grown on cover slips overnight and were washed with PBS, fixed with 1:1 mixture of methanol and acetone, stained with 4',6-diamino-2-phenylindole (DAPI; final concentration 10 µg/ml) and mounted on clean glass slides using 1–2 µl of glycerol. The cells were visualized under confocal microscope (Zeiss LSM 510) and the localization of GFP tagged and DsRed tagged proteins were observed by exciting at 488 and 543 nm, respectively. Subsequent fluorescence was monitored at 503 nm and 583 nm respectively using LSM 510 software.

Sub-cellular fractionation, immunoprecipitation and western blot analysis
Cells grown in 100 mm Petri dishes were washed with ice cold PBS and harvested at 300g for 3 min at 4°C. The pellet was suspended in cytosol extraction buffer (50 mM Tris–Cl pH 7.5, 10 mM NaCl, 2 mM EDTA, 1 mM PMSF and 1x protease inhibitor cocktail) and kept on ice for 15–20 min. Cells were then lysed by adding 0.25% NP-40 and centrifuged immediately at 800g for 5 min at 4°C. The supernatant was kept as cytosolic extract. The pellet was then suspended in nuclear extraction buffer (50 mM Tris–Cl pH 7.5, 400 mM NaCl, 2 mM EDTA, 1 mM PMSF and 1x protease inhibitor cocktail) and kept on ice for 40 min followed by centrifugation at 13 000g for 20 min at 4°C. The supernatant was kept as nuclear extract. For immunoprecipitation assay with cytosolic and nuclear extract, cytosolic extract was prepared as described above. Nucleus was isolated by resuspending the nuclear pellet in nuclear IP buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 2 mM EDTA, 1 mM PMSF and 1x protease inhibitor cocktail) followed by repeated freezing and thawing and centrifugation at 13 000g for 20 min at 4°C. The extracts are then incubated with anti HIP-1 antibody (1:200 dilutions) for 2 h at 4°C. Next BSA soaked protein-G agarose was added to the reaction mix and incubated over night at 4°C under continuous rotating condition. Next day the immunoprecipitated complex was collected by centrifugation at 1000g for 2 min at 4°C. Beads were washed twice with nuclear IP buffer, once with wash buffer I (50 mM Tris–Cl pH 7.5, 400 mM NaCl, 2 mM EDTA, 1 mM PMSF and 1x protease inhibitor cocktail) and finally with wash buffer II (50 mM Tris–Cl pH 7.5, 400 mM NaCl, 2 mM EDTA, 0.1% Triton X-100, 1 mM PMSF and 1x protease inhibitor cocktail). The bound proteins were extracted from beads by boiling with SDS gel loading buffer and were subjected to western blot using anti HIPPI and anti HIP-1 antibody. The methods used for western blot analysis was essentially the same as described earlier (2).

For immunoprecipitation assay using whole cell extract, cells grown in 10 cm Petri dishes were washed in ice cold PBS and harvested at 300g for 3 min at 4°C. The pellet was suspended in co-immunoprecipitation buffer (50 mM Tris–Cl pH 7.5, 15 mM EDTA, 100 mM NaCl, 0.1% Triton X-100 and PMSF with 100 µg/ml final concentrations), lysed by freezing and thawing and centrifuged at 13 000g for 15 min. The supernatant was collected and protein was estimated. To detect GFP tagged proteins, immunoprecipitation with anti-GFP antibody (1:500 dilutions) was carried out using the protocol described above. The beads were washed several times with co-immunoprecipitation buffer and the precipitated complex was extracted from beads by boiling with SDS gel loading dye. Western blot analysis was performed using anti-GFP, anti-caspase-8 and anti-HIP-1 antibodies. For other proteins, western blot analysis was carried out using anti HIP-1; anti HIPPI, anti-GFP, anti-H2B, anti Lamin B and anti Beta actin antibodies after standardizing for appropriate dilutions. Beta actin was used as internal control for cytoplasmic extract while H2B or lamin B was used for loading controls for proteins in the nuclear fractions. Each experiment was repeated 2–3 times. Integrated optical density (IOD) of each band was calculated using Image Master VDS software (Amarsham Biosciences, UK). When ever necessary, IOD was normalized with that of the loading control.

Immunocytochemistry
Cells were grown on cover slips and transfected with respective constructs whenever necessary as described above. Cells (after 24 h of transfection, for transfected cells) were fixed with 3.7% freshly prepared Paraformaldehyde for 20 min at room temperature, washed thrice with PBS, and permeabilized with 0.1% Triton X-100 in PBS for 10 min at room temperature. It was then rinsed thrice with PBS and blocked in 2% BSA for 1 h. The coverslips containing cells were then incubated for 1 h at 37°C with primary antibodies (anti HIP-1, anti HIPPI) with (1:50) dilutions. Cells were then washed thrice with PBS and incubated with fluorophore-conjugated (FITC or TRITC) secondary antibodies at 37°C in dark. Cover slips were again washed with PBS and then mounted on slides. Photographs were taken using (ZEISS LSM 510) confocal microscope.

Chromatin immunoprecipitation (ChIP) and re-chromatin immunoprecipitation (reChIP) assay
Methods used for the ChIP experiments were adapted from the results published earlier (2). GFP-wpDED or GFP-mpDED transfected HeLa cells and K562 were grown on petri plates to 80–90% confluency as mentioned above. Cells were incubated with 2% formaldehyde for 2 min at room temperature to cross-link the proteins with the DNA. This cross-linking reaction was stopped using 150 mM Glycine. Cells were scraped and spun down at 300 g for 2 min, washed twice with PBS and the pellet was frozen in dry ice for 20 mins. Buffer C (20 mM HEPES (pH 7.9), 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA) with 1 mM PMSF was added to the pellet after thawing at 4°C to lyse the cells. Nuclei were spun down at 15 000 g for 10 min and the pellet was re-suspended in breaking buffer (50 mM Tris–HCl pH 8.0, 1 mM EDTA, 150 mM NaCl, 1% SDS and 2% Triton X-100) and sonicated twice (two pulses of 10 s each). Contents were then spun down. The pellet was discarded. Triton buffer (50 mM Tris–HCl pH 8.0, 1 mM EDTA, 150 mM NaCl and 0.1% Triton X-100) was added to the supernatant (nuclear extract). Anti-GFP (for GFP-pDED transfected HeLa cells) or anti-HIPPI (for K562 cell) antibody was added to a part of the nuclear extract (+Ab) and antibody reaction was carried out overnight at 4°C. Other part of the nuclear extract was kept at 4°C (–Ab). The next day Protein G Agarose beads were added to the +Ab and –Ab fractions and left on a shaker for 6 h at 4°C. After 6 h, beads were washed 4 times with Triton buffer and two times with Tris buffer [10 mM Tris–HCl (pH 8.0)].

Next SDS–NaCl–DTT buffer (62.5 mM Tris–HCl, pH 6.8, 200 mM NaCl, 2% SDS 10 mM DTT) was added to the beads and incubated at 65°C overnight for reverse cross-linking. Next day, all the fractions were subjected to phenol chloroform extraction and the aqueous layer was collected. Cross-linked DNA was precipitated with 3 M Sodium acetate and ethanol. DNA pellet was washed with 70% ethanol, dried and then dissolved in distilled water. The DNA so obtained was amplified by PCR using caspase-1 717 bp upstream sequences (4). The primer sequences (5'–3') were as follows:

Forward: 5'-GGAAGATCTGGCTTTTCTCTCTCCCTTC-3'

Reverse: 5'-CGGGGTACCAAGCCTAGGAAACACAAGGAGA-3'

For re-ChIP assay, HeLa cells grown in 10 cm Petri dishes to ~80–90% confluency were transfected with GFP-Hippi construct. After 24 h, transfection efficiency was monitored by fluorescence microscopy and was ~80%. The cells were then cross-linked and the first immunoprecipitation using anti HIPPI antibody was carried out as described above. The immunoprecipitated chromatin was then eluted from beads using ChIP elution buffer (50 mM Tris–HCl, pH 7.5, 10 mM EDTA, 1% SDS) and incubating at 68°C for 10 min. One part of the eluted chromatin was subjected to reverse cross-linking followed by phenol chloroform isolation of DNA as mentioned above to check the first immunoprecipitation. The other part of the eluted DNA-protein complexes was diluted with Triton buffer and incubated with anti HIP-1 antibody over night at 4°C. Next day, the DNA-protein-antibody complex was immunoprecipitated by adding Protein G Agarose beads and DNA was extracted by phenol chloroform isolation followed by ethanol precipitation. PCR amplification of the eluted DNA was carried out using caspase-1 upstream (300 bp) sequence specific primers. The primer sequences (5'–3') were as follows:

Forward: 5'AATGATTGAGAAACTCTTCACTGTGT-3'

Reverse: 5'-CGGGGTACCAAGCCTAGGAAACACAAGGAGA-3'

Real-time PCR
RNA (100 ng) was reverse transcribed as described earlier . Real-time RT–PCR reaction was carried out using Sybr green 2x Universal PCR Master Mix (Applied Biosystems, USA) in ABI Prism 7500 sequence detection system. Each reaction was performed in triplicate using primer sequences for caspase-1 . Non-template control reaction at the same condition was performed to ascertain the baseline and threshold value for the analysis. Ct value obtained for caspase-1 was normalized with respect to beta actin ({Delta}Ct). Differences between the {Delta}Ct values of test and control sets were determined ({Delta}{Delta}Ct). The fold change was determined using the formula 2–{Delta}{Delta}Ct.