Which of the following is required to replicate the lagging strand Which of the following is required to replicate the lagging strand of DNA?
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As the replication fork progresses, synthesis of the discontinuous lagging strand requires frequent priming and cycling of the lagging strand polymerase to the new primers. It appears that this mechanism also permits bypass of template lesions on both strands, leaving the damage behind in a single-strand gap and precluding fork stalling or collapse. Show
Main TextIntroductionThe standard view of DNA replication is that it is semidiscontinuous, with the leading strand synthesized as a single uninterrupted chain and the lagging strand as a series of short (<2 kb) Okazaki fragments. Despite the prevalence of this view and the support it has gained from in vitro studies, it is not in agreement with cytological evidence in E. coli, which routinely shows discontinuities in both strands during DNA replication (reviewed in ). This does not necessarily imply that there is a regular cycle of reinitiation on the leading strand, but it suggests that priming can and does recur on the leading strand as the replication fork moves away from the origin, most likely in response to damage on the template DNA molecule. This review describes our present understanding of the process of discontinuous DNA synthesis on the lagging strand. We also discuss new emerging views of replication in which differences in the mechanisms of leading and lagging strand synthesis are blurred by the need to deal with ever-present lesions on both template strands. Replisome Dynamics in E. coliNumerous different proteins act together to advance a DNA replication fork. In aggregate, these diverse protein actors are referred to as a “replisome,” and the structure and function of the E. coli replisome are illustrated in Figure 1. Parental duplex DNA is unwound by the homohexameric DnaB helicase as it translocates along the lagging strand template in the 5′ to 3′ direction just ahead of the leading strand polymerase (Figure 1A). The helicase activity of DnaB is greatly stimulated by interaction with the τ subunit of DNA polymerase III holoenzyme ( ). DNA polymerase III holoenzyme is a multiprotein complex comprised of two heterotrimeric Pol III core polymerases (αɛθ subunits), two homodimeric β sliding clamp processivity factors, and a single γ complex clamp loader (γ1τ2δδ′χψ) (reviewed in ). The C termini of the τ subunits protrude from the γ complex clamp loader and bind two separate assemblies of Pol III core. These connections enable the clamp loader to serve as a structural bridge between the replicative helicase and the leading and lagging strand polymerases at the prow of the replication fork.  Figure 1The Cycle of Lagging Strand Synthesis on an Undamaged Template Show full caption (A) As the lagging strand polymerase synthesizes an Okazaki fragment, the clamp loader opens a new clamp and the helicase recruits primase to the replication fork to initiate the next fragment. (B) After synthesis of the RNA primer, the clamp loader displaces primase and loads the clamp onto the new primer/template junction. (C) Completion of Okazaki fragment synthesis triggers recycling of the lagging strand polymerase to the newly loaded clamp, leaving the old clamp behind. (D) The lagging strand polymerase synthesizes the new Okazaki fragment, completing a full cycle. Fork unwinding and leading strand synthesis continue throughout the cycle. The authors are grateful to Dr. Nina Yao for the artwork.
Cellular DNA polymerases cannot initiate synthesis in the absence of a nucleic acid primer, so the first step in DNA synthesis is the formation of a short RNA primer (∼10 nt) by specialized RNA polymerases known as primases ( ). In principle, leading strand synthesis requires only a single priming event, whereas frequent repriming is the hallmark of discontinuous lagging strand synthesis. The distribution of primers, ∼1–2 kb apart on the lagging strand, is governed by dynamic interactions between DnaB and the DnaG primase ( ) and possibly, according to a recent report, by interactions between separate primase molecules bound to a single DnaB on the lagging strand ( ). Primase remains bound to the 3′ terminus of the RNA primer through contact with SSB, the single-strand DNA (ssDNA) binding protein, which binds and protects ssDNA ahead of the primer (Figure 1A). The Pol III core polymerase then replaces primase at the primer terminus in a three-part switch activated by the χ subunit of the γ complex clamp loader ( ). χ displaces primase by competitive binding to SSB, whereas the clamp-loading subunits of γ complex (the AAA+ proteins γ, τ2, δ, and δ′) form a helical structure that completely encases the newly cleared primer-template junction (for a detailed review of clamp loaders and how their structures confer specificity for the primed site, see ). In the second part of the switch, the clamp loader binds ATP, opens the ring-shaped β clamp, and positions it around the RNA-DNA duplex (Figure 1B). ATP hydrolysis ejects the clamp loader from the primer-template junction and releases the clamp, allowing it to close around the duplex (Figure 1C) ( Ason et al., 2003
Mechanism of loading the Escherichia coli DNA polymerase III beta sliding clamp on DNA. Bona fide primer/templates preferentially trigger the gamma complex to hydrolyze ATP and load the clamp.
). Pol III core then binds the β clamp to complete the switch and begin synthesis of a new Okazaki fragment (Figure 1D). When bound to Pol III core, the β ring slides along the DNA with the polymerase, converting it into a highly processive enzyme capable of extending DNA chains over 50 kb without dissociating. The E. coli replication fork moves about 1 kb per second under normal circumstances. Okazaki fragments are 1–2 kb in E. coli, so new primers must be synthesized on the discontinuous lagging strand every few seconds. The clamp loader is capable of rapid and repeated loading of new β dimers onto primed sites as they are generated, so this is not a limiting step, but a longstanding conundrum in lagging strand synthesis has been to rationalize how the lagging strand polymerase lets go of DNA after it completes an Okazaki fragment (Figures 1B and 1C). The interaction between β and Pol III core on DNA is very tight, with a dissociation half-life of more than 5 min when bound to the primer-template junction. With only 10–20 molecules of polymerase in the cell, and new Okazaki fragments being produced every few seconds, the lagging polymerase must be used repeatedly. But how does the lagging strand Pol III core rapidly disconnect from the ring-shaped clamp when it finishes a fragment? So far, two mechanisms have been described for disconnecting Pol III core from β on the lagging strand. One of these is an intramolecular signaling process within the Pol III holoenzyme that is triggered when Pol III core finishes an Okazaki fragment and encounters the 5′ terminus of a previous fragment. Often referred to as the “collision mechanism,” this intramolecular signal is mediated by the τ subunit of γ complex, which attenuates the strength of the β-Pol III core interaction and reduces the dissociation half-life from 5 min to far less than 1 s ( , ). This frees the lagging strand DNA polymerase from β and allows it to cycle to the new primer-template junction to begin synthesis of the next Okazaki fragment (Figure 1C). A similar mechanism has been shown to function during replication of bacteriophage T4 ( ). When the lagging strand Pol III core cycles to a new primer, the β clamp is left behind on the completed DNA molecule. β binds to many other proteins, including DNA polymerase I and DNA ligase, and may coordinate their actions in removing RNA primers and covalent joining of the completed Okazaki fragments into a continuous DNA molecule ( ). An alternative mechanism involves premature release of the polymerase, which disengages from its clamp before Okazaki fragment synthesis is complete ( ). A recent study suggests that the premature release mechanism may be common during bacteriophage T4 replication ( ). The authors propose that the signal for premature release is the assembly of a new clamp on an upstream primer; however, this is difficult to prove conclusively, as a clamp must be loaded on the primer in order to obtain an extension signal in the first place. The relative contribution of the collision and premature release mechanisms to ordinary cycling of the lagging strand polymerase remains to be determined, but it seems likely that the ability of the polymerase to release from its clamp prematurely is an important means of keeping the replication fork moving in the face of template damage, as described below. Replisomes Bypass Template DamageOne of the primary constraints on the ability of bacteria to multiply in nutrient-rich environments is the rate at which their genomes can be duplicated. E. coli is capable of reproducing its 4.6 megabase genome from a single origin of replication with high fidelity in less than 40 min, a rate that exerts tremendous selective pressure on the replication machinery to keep the fork moving as quickly as possible. Based on cytological evidence, it was originally assumed that replication skipped over template lesions leaving gaps (see references in ). However, early in vitro studies of T7 and T4 phage replisomes suggested that synthesis of the two strands was tightly coupled and that a block to one polymerase would halt the other polymerase until repair was complete ( , ). More recent studies of the E. coli and T4 replication forks have revealed that conditions that stall the lagging strand polymerase do not stop the leading strand polymerase ( , , , ). Instead, leading and lagging strand synthesis become uncoupled even though the two polymerases remain connected to the γ complex through their respective τ subunits. Continued fork advance provides ssDNA for further priming events, and premature release of the stalled lagging strand polymerase from its β clamp cycles the polymerase to a new primer, restarting lagging strand synthesis and leaving behind an ssDNA gap with a template lesion (Figure 2A). Premature release of polymerase, which is critical to the functional uncoupling of physically linked polymerases, allows continued movement of the replication fork and likely underlies observations of continued leading and lagging strand polymerization in vitro even when complete extension of Okazaki fragment synthesis is prevented by primase starvation ( ) or nucleotide depletion ( ). Figure 2Replisomes Bypass Template Damage Show full caption (A) Upon encountering a lesion on the lagging strand template (stop sign), leading strand synthesis continues and the stalled lagging strand polymerase recycles (dotted arrow) to a new primer/template junction, leaving a single-strand gap with a template lesion (bottom). (B) The leading strand polymerase stalls upon encountering a lesion (top). The helicase recruits primase to reinitiate leading strand synthesis ahead of the lesion, leaving a single-strand gap (bottom). If stalling causes the replication fork to collapse, additional factors (e.g., PriA or PriC) are required to reload the helicase at the collapsed fork. For (A) and (B), gaps that are left behind in either strand can be repaired with high fidelity by recombination processes using the new sister chromatid as a template. Artwork by Dr. Nina Yao.
Interestingly, DNA damage is also observed to result in ssDNA gaps in the leading strand of both prokaryotes (reviewed in ) and eukaryotes ( ), suggesting that the fork may circumvent lesions on the leading strand in a similar way. Underlying the ability of the lagging strand machinery to skip past DNA damage is the fact that new RNA primers are regularly available for reinitiating upstream synthesis on the lagging strand. Primase requires interaction with the DnaB helicase to initiate RNA synthesis, and because DnaB is located on the lagging strand, it was thought that primase could not act on the leading strand. However, a surprising new finding shows that primase can form new primers on the leading strand by binding the DnaB helicase on the lagging strand ( ). This allows the replication fork to continue past damaged sites in the leading strand template, leaving the lesion behind in an ssDNA gap (Figure 2B). Further study will be needed to determine whether the same premature release mechanism used on the lagging strand is also operative on the leading strand or whether it occurs through a different process. The recent work in E. coli reveals that replisomes are exceedingly efficient in moving forward, simply jumping over template lesions as they are encountered. At first glance, it may seem rather slipshod for replication forks to bypass DNA damage in this way. However, by leaving the lesion behind in a ssDNA gap, the fork can proceed while the lesion is repaired by the high-fidelity RecA-mediated recombinational repair system (reviewed in ). Previous models for repair of forks stalled at lesions required recombination-mediated fork regression in a complex series of DNA transactions. Implicit in those models is the idea that replication forks collapse, stopping replication and requiring reassembly of new replisomes. The ability of replisomes to prime past lesions and bypass them precludes the requirement for fork regression to repair the lesion. It should be noted, however, that replisome encounter with a nicked template is still expected to involve complete collapse of the replication fork. Can the replisome bypass all forms of template damage? The profusion of translesion DNA polymerases in the cell (especially in eukaryotes) indicates that some lesions may prevent movement of the fork altogether. Indeed, both the high-fidelity Pol III and low-fidelity Pol IV (in the DinB/Y family) bind the β clamp simultaneously as it moves along DNA, allowing for efficient switching between replication and repair modes in the face of template damage (reviewed in ). These finely tuned dynamics between a replisome and a low-fidelity bypass polymerase on one sliding clamp imply that in some instances the replication apparatus deals with template lesions directly. Eukaryotic ReplicationAll of the functional complexes required to replicate the E. coli genome have identifiable counterparts in eukaryotic cells (Table 1), and most of these have been purified and characterized, some extensively. Although the ability of these key components to support replication in the SV40 system was identified several years ago (reviewed by ), the goal of reconstituting a fully operational eukaryotic replication fork in vitro remains elusive. As with most biological processes, eukaryotic replication requires additional proteins with no eubacterial homolog (Table 1), but the functional roles of these essential factors are still uncertain ( ). Table 1Key Components of the Replisome
a Pol δ is a heterotrimer in S. cerevisiae. b In E. coli, the clamp loader consists of five subunits (γτ2δδ′) plus χ and ψ, which have auxiliary roles.
One of the most basic questions about eukaryotic replication, whether there are separate polymerases for the leading and lagging strands, still does not have a satisfactory answer. Although the likely replicative polymerases, Pol δ and Pol ɛ, are both essential for cell viability, the catalytic activity of Pol ɛ is dispensable, suggesting that DNA synthesis is not its essential function ( ). On the other hand, immunodepletion experiments in Xenopus egg extracts showed an approximately equivalent reduction in the total amount of DNA synthesis in extracts depleted of either Pol δ or Pol ɛ ( ). Pol ɛ was originally identified on the basis of a repair assay ( ), and therefore, this polymerase may play dual roles in the cell. As the roles of Pol δ and Pol ɛ are revealed through further genetic and biochemical studies, one of the most interesting questions will be whether the leading and lagging strand polymerases are physically connected as in E. coli. There is no known counterpart to the τ C-terminal extension among the eukaryotic RFC subunits, so if there is an analogous structural link between the replicative helicase and one or both of the polymerases it remains to be identified. A recent report showing a direct interaction between archaeal homologs of the eukaryotic GINS and MCM complexes suggests that GINS might serve as a bridge between the replicative helicase and other components of the fork, possibly including the primase ( ). One area in which our understanding of eukaryotic lagging strand replication is particularly advanced is in the removal of RNA from completed Okazaki fragments and their ligation to the upstream fragment (reviewed in ). Pol δ initiates the process by balancing its polymerase and 3′–5′ proofreading exonuclease functions to regulate displacement of the 5′ end of the downstream Okazaki fragment, thereby converting the downstream RNA primer into an incremental 5′ flap substrate for the Dna2 and Fen1 nucleases ( ). The crystal structure of human DNA ligase I reveals that, like the PCNA clamp to which it binds, ligase encircles the duplex, but unlike PCNA, it makes extensive contact with the DNA to carry out the ligation reaction ( ). Fen1 also binds PCNA, suggesting that the clamp may mediate yet another molecular switch similar to the primase-polymerase switch described above. Concluding RemarksRepeated RNA priming is required for discontinuous synthesis on the lagging strand, and the replisome must act in a highly dynamic fashion to accommodate this mode of synthesis. Replication of the continuous leading strand seems exceedingly simple in comparison. However, recent work in E. coli reveals that, by virtue of its ability to cope with multiple priming events, the replisome has gained an astounding degree of freedom. The regular action of primase on the lagging strand, and incidental repriming ahead of leading strand damage, enables the replisome to skip over replication-blocking lesions and continue on its way. Eukaryotes have numerous extra factors for replication, but it may be presumed that the fundamental features of fork progression discovered in prokaryotic systems will generalize. On the other hand, because eukaryotes typically initiate replication from many origins on each chromosome, failure to complete synthesis from one particular origin may not be problematic, so the need to keep an individual replication fork moving at all costs might not be as great as in E. coli. In any case, there remains a great deal of challenging work to be done in “unwinding” the workings of the replication fork. References
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Related ArticlesWhat is required to replicate the lagging strand of DNA?The other (the lagging strand) is made in small pieces. DNA replication requires other enzymes in addition to DNA polymerase, including DNA primase, DNA helicase, DNA ligase, and topoisomerase.
What is needed for lagging strand?The leading strand is synthesized in a continuous manner, whereas synthesis of the lagging strand requires a primase that makes RNA primers that are extended by the DNA polymerase to form Okazaki fragments — short DNA fragments that are processed to produce a continuous DNA strand.
What does the lagging strand replicate?The lagging strand is a single DNA strand that, during DNA replication, is replicated in the 5′ – 3′ direction (opposite direction to the replication fork). DNA is added to the lagging strand in discontinuous chunks called 'okazaki fragments'.
Which of the following are required for DNA replication?DNA replication requires DNA polymerase and ligase. A molecule of a DNA polymerase binds to one strand of the DNA and begins moving along it in the 3' to 5' direction, using it as a template for assembling a leading strand of nucleotides and reforming a double helix.
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