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.