Dna Replication

DNA REPLICATION General Features of Chromosomal Replication DNA Replication Is Semiconservative

Is replication a conservative or semiconservative process? In the first mechanism, the two new strands form a new duplex and the old duplex remains intact, whereas in the second mechanism, each old strand becomes paired with a new strand copied from it . M. Meselson and W. F. Stahl. experiment E. coli cells initially were grown in a medium containing ammonium salts prepared with "heavy" nitrogen (15N) until all the cellular DNA contained the isotope. The cells were then transferred to a medium containing the normal "light" isotope (14N), and samples were removed periodically from the cultures. The DNA in each sample was analyzed by density-gradient equilibrium centrifugation, which can separate heavy-heavy (H-H), light-light (L-L), and heavy-light (H-L) duplexes into distinct bands. Apparently all cellular DNA in both prokaryotic and eukaryotic cells is replicated by a semiconservative mechanism.

Conservative and semiconservative mechanisms of DNA replication differ in whether the newly synthesized strands pair with each other (conservative) or with an old strand (semiconservative).

The Meselson Stahl Experiment : What is the mode of DNA replication?

The Meselson-Stahl experiment a) Cells were grown for many generations in a medium containing only heavy nitrogen, 15N, so that all the nitrogen in their DNA was 15N, as shown by a single band (blue) when centrifuged in a CsCl density gradient b) Once the cells had been transferred to a medium containing only light nitrogen, 14N, cellular DNA isolated after one generation equilibrated at a higher position in the density gradient (purple band). c) Continuation of replication for a second generation yielded two hybrid DNAs and two light DNAs (red), confirming semiconservative replication.

Most DNA Replication Is Bidirectional

Three mechanisms of DNA strand growth that are consistent with semiconservative replication 1. One new strand derives from one origin and the other new strand derives from another origin. Only one strand of the duplex grows at each growing point. Operates in linear DNA viruses such as adenovirus, the ends of the DNA molecules serve as fixed sites for the initiation and termination of replication. 2. One origin and one growing fork (the point where DNA replication occurs), which moves along the DNA in one direction with both strands of DNA being copied). Certain bacterial plasmids replicate in this manner. 3. Synthesis might start at a single origin and proceed in both directions, so that both strands are copied at each of two growing forks. Common in prokaryotic and eukaryotic cells: that is, DNA replication proceeds bidirectionally from a given starting site, with both strands being copied at each fork. Thus two growing forks emerge from a single origin site.

DNA Replication Begins at Specific Chromosomal Sites

Most important decision every cell has to make is whether, and when, to replicate its DNA.

DNA replication is controlled at the initiation step. Control would be most efficient if there are specific sites on chromosomes at which DNA replication always begins in vivo. Molecular studies indicate that replication of these DNAs actually begins at a defined sequence of base pairs near the center of these bubbles, called the replication origin. Replication origin - Stretch of DNA that is necessary and sufficient for replication of a circular DNA molecule, usually a plasmid or virus, in an appropriate host cell. E. coli Replication Origin

The E. coli replication origin oriC is an ≈240-bp DNA segment present at the start site for replication of E. coli chromosomal DNA. Plasmids or any other circular DNA containing oriC are capable of independent and controlled replication in E. coli cells. oriC contain repetitive 9-bp and AT-rich 13-bp sequences, referred to as 9-mers (dnaA boxes) and 13-mers, respectively, these are binding sites for the DnaA protein that initiates replication. SV40 Replication Origin

A 65-bp region in the SV40 chromosome is sufficient to promote DNA replication both in animal cells and in vitro. Three Common Features of Replication Origins

1. Replication origins are unique DNA segments that contain multiple short repeated sequences. 2. These short repeat units are recognized by multimeric origin-binding proteins. These proteins play a key role in assembling DNA polymerases and other replication enzymes at the sites where replication begins. 3. Origin regions usually contain an AT-rich stretch. This property facilitates unwinding of duplex DNA because less energy is required to melt A·T base pairs than G·C base pairs . Origin-binding proteins control the initiation of DNA replication by directing assembly of the replication machinery to specific sites on the DNA chromosome.

DnaA Protein Initiates Replication in E. coli

Genetic studies first suggested that initiation of replication at oriC most likely depended on the protein encoded by a gene designated dnaA. Initially, mutant strains carrying temperature-sensitive mutations in dnaA were isolated; these cells grew at permissive temperatures (e.g., 30 °C) but not at nonpermissive temperatures (39 42 °C). When E. coli cells carrying such conditional lethal mutations had begun DNA replication at the permissive temperature and then were shifted to the higher temperature, they completed the round of DNA synthesis already under way; however, they did not start another round of replication at the nonpermissive temperature. Subsequent genetic studies with recombinant E. coli further pinpointed the DnaA protein as a prime candidate for interaction with oriC.

In vitro studies showed that pure DnaA protein binds to the four 9-mers in oriC, forming an initial complex that contains 10 20 protein subunits. Furthermore, although DnaA can bind to duplex E. coli origin DNA in the relaxed-circle form, it can initiate replication only if the DNA is negatively supercoiled. The reason for this specificity is that DNA molecules with negative supercoils are tightly wound and are easier to melt locally (thus providing a single-stranded template region) than are DNA molecules without supercoils. Supercoiling of DNA and the enzymes that control the degree of DNA supercoiling, called topoisomerases, are discussed in detail later. Binding of DnaA to the oriC 9-mers facilitates the initial strand separation, or "melting," of E. coli duplex DNA, which occurs at the oriC 13-mers. This process requires ATP and yields a so-called open complex. When a mixture of E. coli DNA and DnaA protein is treated with an endonuclease that specifically recognizes single-stranded DNA, the DNA is cut in the origin region, demonstrating that it is melted.

Model of initiation of replication at E. coli oriC

• • •

The 9-mers and 13-mers are the repetitive sequences Multiple copies of DnaA protein bind to the 9-mers at the origin and then "melt" (separate the strands of) the 13-mer segments. The role function of DnaC is to deliver DnaB, which is composed of six identical subunits, to the template.

•

One DnaB hexamer clamps around each single strand of DNA at oriC, forming the prepriming complex. DnaB is a helicase, and the two molecules then proceed to unwind the DNA in opposite directions away from the origin.

DnaB Is an E. coli Helicase That Melts Duplex DNA

Further melting of the two strands of the E. coli chromosome to generate unpaired template strands is mediated by the protein product of the dnaB locus, a helicase that is essential for DNA replication. One molecule of DnaB, a hexamer of identical subunits, clamps around each of the two single strands in the open complex formed between DnaA and oriC. This binding requires ATP and the protein product encoded by the dnaC locus, which "escorts" DnaB to the DnaA proteins, yielding the prepriming complex (see Figure 12-7). Helicases constitute a class of enzymes that can move along a DNA duplex utilizing the energy of ATP hydrolysis to separate the strands. In E. coli, the separated strands are inhibited from subsequently reannealing by a single-strand-binding protein (SSB protein), which binds to both separated strands. When temperature-sensitive dnaB mutants are shifted to nonpermissive temperatures, unwinding ceases; as a result, DNA synthesis stops immediately for lack of singlestranded templates. Helicases like DnaB bind to a single-stranded segment of DNA, then move along that strand melting the hydrogen bonds that link it to its complementary strand. Like many proteins that bind to DNA, helicases exhibit a directionality with respect to the unwinding reaction. DnaB moves along the single strand of DNA to which it binds in the direction of its free 3 end, and in this sense it is said to unwind DNA in the 5 → 3 direction (Figure 12-8). DnaB, like many other proteins that act on DNA, is processive. Because it forms a clamp around a single strand of DNA, DnaB does not "fall off" until it reaches the end of that strand or is "unloaded" from DNA by another protein. Other kinds of DNA helicases unwind in the opposite direction, moving along the strand to which they are bound toward the free 5 end.

Helicase activity of E. coli DnaB protein • • •

In the presence of ATP and single-strand-binding (SSB) protein, purified DnaB can unwind a gapped DNA duplex in vitro. Unwinding occurs predominantly in the direction of the 3 end of the strand to which a DnaB molecule is attached. This strand acts as the template for synthesis of the lagging DNA strand. The SSB protein binds to unpaired DNA strands and prevents them from reannealing.

E. coli Primase Catalyzes Formation of RNA Primers for DNA Synthesis

As noted earlier, DNA polymerases can only elongate existing primer strands of DNA or RNA. The primers used during DNA replication in both prokaryotes and eukaryotes are short RNA molecules whose synthesis is catalyzed by the RNA polymerase primase. E. coli strains with temperaturesensitive mutations in dnaG, which encodes primase, cannot replicate their DNA at the nonpermissive temperature, thereby establishing the essential role of primase. Primase is usually recruited to a segment of single-stranded DNA by first binding to a DnaB hexamer already attached at that site. The term primosome is now generally used to denote a complex between primase and helicase, sometimes with other accessory proteins. In initiation of E. coli DNA replication, a primosome is formed by binding of primases to DnaB in the prepriming complex (see Figure 12-7). After the bound primases synthesize short primer RNAs complementary to both strands of duplex DNA, they dissociate from the single-stranded template.

At a growing fork, one strand is synthesized from multiple primers (a) The overall structure of a growing fork. Synthesis of the leading strand, catalyzed by DNA polymerase III, occurs by sequential addition of deoxyribonucleotides in the same direction as movement of the growing fork. (b) Steps in the discontinuous synthesis of the lagging strand. This process requires multiple primers, two DNA polymerases, and ligase, which joins the 3 hydroxyl end of one (Okazaki) fragment to the 5 -phosphate end of the adjacent fragment. (c) DNA ligation. During this reaction, ligase transiently attaches covalently to the 5 phosphate of one DNA strand, thus activating the phosphate group.

Figure 12-10. A β -subunit dimer tethers the core of E. coli DNA polymerase III to DNA, thereby increasing its processivity. (a) Space-filling model based on x-ray crystallographic studies of the dimeric β subunit binding to a DNA duplex. Two β subunits (red and yellow) form a donut-like clamp that remains tightly bound to a closed circular DNA molecule, but readily slides off the ends of a linear DNA molecule. (b) Schematic diagram of proposed association of the core polymerase (green) with the β subunit clamp at the primer-template terminus. This interaction keeps the core from "falling off" the template and positions it near the point of nucleotide addition, increasing the processivity of the core polymerase by more than a thousandfold.

The Leading and Lagging Strands Are Synthesized Concurrently

Once the prepriming complex and an RNA primer are formed at the E. coli replication origin, chain elongation to yield the leading strand proceeds with little difficulty. As we've seen, however, lagging-strand synthesis proceeds discontinuously from multiple primers. Two molecules of core DNA polymerase III are bound at each growing fork; one adds nucleotides to the leading strand,

and the other adds nucleotides to the lagging strand. Coordination between elongation of the leading and lagging strand is essential; otherwise one template strand would be incorporated into a duplex with a newly synthesized complementary strand while large parts of the other template strand would remain single-stranded. Figure 12-11 shows how this coordination is achieved. The two core-polymerase molecules at the fork are linked together by a τ -subunit dimer. The core polymerase synthesizing the leading strand moves, together with its β -subunit clamp, along its template in the direction of the movement of the fork, elongating the leading strand. It follows closely the movement of the DnaB helicase bound to the lagging-strand template as the helicase melts the duplex DNA at the fork. Since this corepolymerase molecule remains attached to the DNA template, leading-strand synthesis occurs continuously. The other core-polymerase molecule, which elongates the lagging strand, moves with its β -subunit clamp in the direction opposite to that of the fork movement. As elongation of the lagging strand proceeds, the size of the DNA "loop" between this core polymerase and the fork increases. One way to see this is to imagine the core 2 polymerase fixed in space, linked to core 1; double-stranded DNA newly synthesized by core 2 would be "pushed" into the loop. Eventually the core polymerase synthesizing the lagging strand will complete an Okazaki fragment; it then dissociates from the DNA template, but the τ -subunit dimer continues to tether it to the forkprotein complex. Simultaneously, a primase binds to a site adjacent to the DnaB helicase on the single-stranded segment of the lagging-strand template and initiates synthesis of another RNA primer. The resulting DNA-primer complex attracts another β clamp to this segment of the lagging-strand template, followed by re-binding of the core polymerase, which is still attached to the fork complex. This polymerase molecule then proceeds to elongate the RNA primer to form another Okazaki fragment. As mentioned earlier, as each Okazaki fragment nears completion, the RNA primer of the previous fragment is removed by the 5 3 exonuclease activity of DNA polymerase I. This enzyme also fills in the gaps between the lagging-strand fragments, which then are ligated together by DNA ligase (see Figure 12-9b). Although the two core polymerase molecules are linked by the τ -subunit dimer, they are oriented in opposite directions (see Figure 12-11). Thus, the 3 growing ends of both the leading and lagging strands are close together but offset from each other. For this reason, the point in the template at which the lagging strand is being copied is displaced from the point in the template at which leading-strand copying is occurring. Nonetheless, the two core polymerases can add deoxyribonucleotides to the growing strands at the same time and rate, so that leading- and laggingstrand synthesis occurs concurrently. One τ subunit also contacts the DnaB helicase at the fork. Experiments with purified replication proteins have shown that this interaction increases the normally slow unwinding rate of the helicase ( 35 bp/s) over tenfold, thereby enabling the fork to move at rates up to 1000 bp/s. Thus, there is a physical and functional link between the two major replication machines at the fork the two core polymerases and the primosome complex of primase and DnaB. By closely coordinating all the events depicted in Figures 12-9 and 12-11, the growing fork moves 500 1000 bp/s while both strands are being replicated.

Schematic model of the relationship between E. coli replication proteins at a growing fork A single DnaB helicase moves along the lagging-strand template toward its 3 end, thereby melting the duplex DNA at the fork. 2. One core polymerase (core 1) quickly adds nucleotides to the 3 end of the leading strand as its single-stranded template is uncovered by the helicase action of DnaB. This leading-strand polymerase, together with its β -subunit clamp, remains bound to the DNA, synthesizing the leading strand continuously. 3. A second core polymerase (core 2) synthesizes the lagging strand discontinuously as an Okazaki fragment. The two core polymerase molecules are linked via a dimeric τ protein. 4. As each segment of the single-stranded template for the lagging strand is uncovered, it becomes coated with SSB protein and forms a loop. Once synthesis of an Okazaki fragment is completed, the lagging-strand polymerase dissociates from the DNA but the core remains bound to the τ -subunit dimer. The released core polymerase subsequently rebinds with the assistance of another β clamp in the region of the primer for the next Okazaki fragment. 1.

At a Growing Fork One Strand Is Synthesized Discontinuously from Multiple Primers

We have seen how the activities of helicase and primase solve two of the problems inherent to DNA replication unwinding of the duplex template and the requirement of DNA polymerases for a primer. Remember, though, that both strands of the DNA template are copied as the replication bubble enlarges. Each end of the bubble represents a growing fork where both new strands are synthesized (see Figure 12-2c). At each growing fork, one strand, called the leading strand, is synthesized continuously from a single primer on the leading-strand template and grows in the 5 → 3 direction. Growth of the

leading strand proceeds in the same direction as movement of the growing fork (Figure 12-9a). Synthesis of the lagging strand is more complicated, because DNA polymerases can add nucleotides only to the 3 end of a primer or growing DNA strand. Movement of the growing fork unveils the template strand for lagging-strand synthesis in the 5 → 3 direction; thus the overall direction of growth of the lagging strand must be from its 3 end toward its 5 end, complementary to the polarity of its template but opposite to the direction of nucleotide addition by DNA polymerases. In both prokaryotes and eukaryotes these apparently incompatible requirements are met by the discontinuous copying of the lagging strand from multiple primers, a process involving several steps. As synthesis of the leading strand progresses, sites uncovered on the single-stranded template of the lagging strand are copied into short RNA primers (<15 nucleotides) by primase (Figure 12-9b). Each of these primers is then elongated by addition of deoxyribonucleotides to its 3 end. In E. coli, this reaction is catalyzed by DNA polymerase III (Pol III), one of three DNA polymerases produced by E. coli. Thus each lagging strand grows in a direction opposite to that in which the growing fork is moving. The resulting short fragments, containing RNA covalently linked to DNA, are called Okazaki fragments, after their discoverer Reiji Okazaki. In bacteria and bacteriophages, Okazaki fragments contain 1000 2000 nucleotides, and a cycle of Okazaki-strand synthesis takes about 2 seconds to complete. In eukaryotic cells, Okazaki fragments are much shorter (100 200 nucleotides). As each newly formed segment of the lagging strand approaches the 5 end of the adjacent Okazaki fragment (the one just completed), E. coli DNA polymerase I takes over. Unlike polymerase III, polymerase I has 5 → 3 exonuclease activity, which removes the RNA primer of the adjacent fragment; the polymerization activity of polymerase I simultaneously fills in the gap between the fragments by addition of deoxyribonucleotides. Finally, another critical enzyme, DNA ligase, joins adjacent completed fragments (Figure 12-9c). E. coli DNA Polymerase III Catalyzes Nucleotide Addition at the Growing Fork

Three DNA polymerases (I, II, and III) have been purified from E. coli (Table 12-1). In addition to its role in filling the gaps between Okazaki fragments, DNA polymerase I probably is the most important enzyme for gap filling during DNA repair. DNA polymerase II functions in the inducible SOS response discussed later; this polymerase also fills gaps and appears to facilitate DNA synthesis directed by damaged templates. Our discussion here focuses on DNA polymerase III, which catalyzes chain elongation at the growing fork in E. coli. The DNA polymerase III holoenzyme is a very large (>600 kDa), highly complex protein composed of 10 different polypeptides. The so-called core polymerase is composed of three subunits. The α subunit contains the active site for nucleotide addition, and the ε subunit is a 3 → 5 exonuclease that removes incorrectly added (mispaired) nucleotides from the end of the growing chain. (This "proofreading" activity of DNA polymerase III is described later.) The function of the Θ subunit is not known. The central role of the remaining subunits is to convert the core polymerase from a distributive enzyme, which falls off the template strand after forming relatively short stretches of DNA containing 10 50 nucleotides, to a processive enzyme, which can form stretches of DNA containing up to 5 × 105 nucleotides without being released from the template. This latter activity is necessary for efficient synthesis of both leading and lagging strands. The key to the processive

nature of DNA polymerase III is the ability of the β subunit to form a donut-shaped dimer around duplex DNA and then associate with and hold the catalytic core polymerase near the 3 terminus of the growing strand (Figure 12-10). Once tightly associated with the DNA, the β -subunit dimer functions like a "clamp," which can slide freely along the DNA, like a ring on a string, as the associated core polymerase moves. In this way, the active sites of the core polymerase remain near the growing fork and the processivity of the enzyme is maximized. Remarkably, of the six remaining subunits, five (γ , δ , δ , χ , and ψ ) form the so-called γ complex, which mediates two essential tasks: (1) loading of the β -subunit clamp onto the duplex DNA primer substrate in a reaction that requires hydrolysis of ATP and (2) unloading of the β -subunit clamp after a strand of DNA has been completed. Loading and unloading of the β -subunit clamp requires opening the clamp ring, but exactly how the γ complex accomplishes this feat is not known. The final subunit (τ ) acts to dimerize two core polymerases and, as summarized in the next section, is essential for coordinating the synthesis of the leading and lagging strands at each growing fork.

Eukaryotic Replication Machinery Is Generally Similar to That of E. coli

As in E. coli, researchers investigating DNA replication in eukaryotes initially concentrated on characterizing the different DNA polymerases present in eukaryotic cells (see Table 12-1). This work was followed by development of in vitro systems for copying small chromosomes from animal viruses (e.g., SV40) whose replication is dependent almost entirely on host-cell proteins. As a result of these studies, the SV40 chromosome now can be replicated in vitro using only eight purified components from mammalian cells. The specific functions of these proteins are highly reminiscent of the E. coli proteins required for replication of plasmids carrying oriC. Thus, the mechanistic problems involved in DNA replication, which are similar in all organisms, have been solved in most cases by use of similar types of proteins. Like DNA replication in E. coli, eukaryotic DNA replication occurs bidirectionally from RNA primers made by a primase; synthesis of the leading strand is continuous, while synthesis of the lagging strand is discontinuous. In contrast to the situation in E. coli, however, two distinct DNA polymerases, α and either δ or ε , function at the eukaryotic growing fork. As depicted in Figure 12-12, replication of SV40 DNA is initiated at a unique site, the replication origin, by binding of a virus-encoded protein called T antigen, or Tag (step 1). This multifunctional, site-specific DNA-binding protein locally melts duplex DNA through its helicase activity. Opening of the duplex at the SV40 origin also requires ATP and replication protein A (RPA), a host-cell single-strand-binding protein with a function similar to that of SSB protein in E. coli cells (step 2). One molecule of polymerase α (Pol α ), tightly associated with a primase, then binds to each unwound template strand. The primases form RNA primers, which are elongated for a short stretch by Pol α , forming the first part of the leading strands, which grow from the origins on the two template strands in opposite directions (step 3). The activity of Pol α is stimulated by replication factor C (RFC). PCNA (proliferating cell nuclear antigen) then binds at the primer-template 3 termini, displacing Pol α from both leading-strand templates and thus interrupting leading-strand synthesis (Figure 1212, step 4). Next, Pol δ binds to PCNA at the 3 ends of the growing strands. The association of Pol δ with PCNA increases the processivity of the polymerase, so that it can continue synthesis of the leading strands without further interruption (step 5). The function of PCNA thus is highly analogous

to that of the β -subunit clamp of E. coli polymerase III, as both proteins form similar "rings" through which the DNA slides. However, their amino acid sequences are dissimilar, and the β clamp is a dimer, whereas PCNA is a trimer. As melting of the duplex DNA, catalyzed by a hexameric form of Tag, progresses farther away from the origin, the primase Pol α complex associates with the melted template strands downstream from the leading-strand primers. Synthesis of the lagging strand then is carried out by combined action of primase and Pol α , along with RFC, while leading-strand synthesis on the other side of the origin also proceeds (see Figure 12-12, step 5). Finally, in eukaryotes, as in E. coli, topoisomerases play an important role in relieving torsional stress induced by growing-fork movement and in separating the two daughter chromosomes. Much has been learned about the eukaryotic proteins that can carry out replication of SV40 viral DNA in vitro. As noted above, initiation of SV40 replication in vitro requires the viral protein T antigen. Studies on replication of eukaryotic cellular DNA in vitro have been hampered by the lack of eukaryotic experimental systems that can sustain in vitro replication initiated at cellular origins and by the lack of in vitro replication systems prepared from extracts of genetically tractable organisms such as yeast. As discussed in Chapter 13, the recent identification of a protein complex that binds to yeast chromosomal origins may be an important first step toward detailed research on cellular DNA replication using a combined genetic and biochemical strategy, which has proved so profitable in E. coli.

Model of in vitro replication of SV40 DNA by eukaryotic enzymes 1.

2.

3. 4. 5.

The top strand is the leading-strand template for the leftward-moving fork, and the bottom strand is the leading-strand template for the rightward-moving fork. Initiation begins by binding of T antigen (Tag) and other proteins to the origin which induces local melting of the duplex DNA. This is followed by binding of a hexameric form of T ag to the leading-strand template at each growing fork Functioning as a helicase, each Tag moves along the strand to which it is bound toward the 5 end, melting the double-stranded DNA as it goes; thus Tag helicase and E. coli DnaB move in opposite directions along DNA. Polymerase α (Pol α ), which is tightly associated with a primase, forms the 5 ends of the leading strands and then is displaced from the template. Association of polymerase δ (Pol δ ) with PCNA increases the processivity of this polymerase, so that it can synthesize the remainder of the leading strands Lagging-strand synthesis downstream from the leading-strand primers is thought to be carried out by the combined action of primase and Pol α . RFC stimulates the activity of Pol α .. RPA=replication protein A; RFC=replication factor C; PCNA=proliferating cell nuclear antigen.

Table 12-1. Properties of DNA Polymerases E. coli Polymerization: 5 3 Exonuclease activity: 3

5

5 3 Synthesis from: Intact DNA Primed single strands Primed single strands plus single-strand-binding protein In vitro chain elongation rate (nucleotides per minute) Molecules present per cell Mutation lethal? Mammalian Cells* Polymerization: 5

3

Exonuclease proofreading activity: 3 5 Synthesis from: RNA primer DNA primer Associated DNA primase Sensitive to aphidicolin (inhibitor of cell DNA synthesis) Cell location: Nuclei Mitochondria

I +

II +

III +

+

+

+

? ?

+ 30,000 10 20 +

β +

γ +

δ ε + +

+

+ +

+

+ ? + +

+

+ + 600 400 + α +

+ + + + +

+

+ + +

+ + +

Yeast DNA polymerase I, II, and III are equivalent to polymerase α , β , and δ , respectively. I and III are essential for cell viability. Polymerase β is most active on DNA molecules with gaps of about 20 nucleotides and is thought to play a role in DNA repair. FEN1 is the eukaryotic 5 3 exonuclease that removes RNA primers; it is similar in structure and function to the domain of E. coli polymerase I that contains the 5 → 3 exonuclease activity. *

The Role of Topoisomerases in DNA Replication DNA molecules can coil and bend in space, leading to changes in topology, including formation of negative or positive supercoils.

Local unwinding of a DNA duplex whose ends are fixed causes stress that is relieved by supercoiling. Any enzyme that cleaves only one strand of a DNA duplex and then reseals it is classified as a type I topoisomerase (Topo I). The Topo I from E. coli acts on negative, but not positive, supercoils.

Action of E. coli type I topoisomerase (Topo I) •

•

• • •

E. coli topoisomerase I, can remove negative supercoils without leaving nicks in the DNA molecule. After the enzyme binds to a DNA molecule, it cuts one strand, simultaneously generating a covalent phosphoester bond between the released 5 phosphate on the DNA and a tyrosine residue in the enzyme. Formation of this phosphotyrosine bond does not require ATP or another source of energy. The free 3 -hydroxyl end of the DNA is held noncovalently by the enzyme. The DNA strand that has not been cleaved is then passed through the single-stranded break. The cleaved strand is then resealed, forming a structure with the same chemical bonds as the starting DNA, but with one less negative supercoil. By this mechanism, the enzyme removes one negative supercoil at a time.

Type II Topoisomerases Change DNA Topology by Breaking and Rejoining Double-Stranded DNA

Type II topoisomerase (Topo II) was isolated from E. coli. and named DNA gyrase. Topo II enzymes have the ability to cut both strands of a double-stranded DNA molecule, pass another portion of the duplex through the cut, and reseal the cut in a process that utilizes ATP Depending on the DNA substrate, these maneuvers will have the effect of changing a positive supercoil into a negative supercoil or of increasing the number of negative supercoils by 2. All type II topoisomerases catalyze catenation and decatenation, that is, the linking and unlinking, of two different DNA duplexes DNA gyrase is composed of two identical subunits. Hydrolysis of ATP by gyrase's inherent ATPase activity powers the conformational changes that are critical to the enzyme's operation). The enzyme functions to introduce negative supercoils at or near the oriC site in the DNA template; as noted earlier, DnaA can initiate replication only on a negatively supercoiled template. Replicated Circular DNA Molecules Are Separated by Type II Topoisomerases Linear Daughter Chromatids Also Are Separated by Type II Topoisomerases

Action of E. coli DNA gyrase, a type II topoisomerase a) Introduction of negative supercoils. The initial folding introduces no stable change, but the subsequent activity of gyrase produces a stable structure with two negative supercoils. Eukaryotic Topo II enzymes cannot introduce supercoils but can remove negative supercoils from DNA. b) Catenation and decatenation of two different DNA duplexes. Both prokaryotic and eukaryotic Topo II enzymes can catalyze this reaction.

A model of DNA replication that accounts for a circular DNA molecule producing linear daughter double helices.