Figure 1: Recombination-Dependent Replication and Direct Restart Mechanisms of Reactivating Forks Stalled by Leading-Strand-Specific Lesions--When the replisome encounters a lesion (black triangle) in the leading-strand template, the resulting stalled fork can have a nascent lagging strand that has progressed past the nascent leading strand if the leading- and lagging-strand polymerases become uncoupled. The stalled fork can undergo nascent-strand regression to form a four-way, Holliday junction structure. From this point, the fork could be reactivated by one of several mechanisms. (a) A recombination-dependent replication (RDR) pathway in which the Holliday junction is resolved by the branch-migration helicase/Holliday-junction endonuclease RuvABC. After excision of the template lesion, the broken chromosome can undergo recombination with the intact duplex to form a D-loop structure. Replication restart then takes place. (b) In the template-switching model, the nascent leading strand is extended, using the nascent lagging strand as a template. After reversal back to a fork structure and direct restart on the fork structure, the lesion is bypassed. (c) Nascent-strand regression allows for excision of the template lesion. The regressed DNA of both the nascent leading and lagging strand is degraded by an exonuclease (indicated in grey) and direct replication restart can take place. Black strands, parental DNA; red strands, nascent leading-strand DNA; blue strands, nascent lagging-strand DNA. The arrowheads at the end of the nascent DNA represent the 3'-OH termini.
Figure 2: PriA and PriC Recognize Different Structures for Replication Restart--(A) The replication restart protein PriA targets those DNA structures for replisome reassembly that possess a 3'-OH terminus in close proximity to the branch junction, such as a D-loop, an R-loop or a stalled fork that contains nascent leading-strand DNA. Replication restart can take place by recombination-dependent replication (RDR) or by a direct restart mechanism. (B) By contrast, PriC-dependent direct restart works most efficiently on stalled forks with a large single-stranded DNA gap, in which lagging-strand DNA synthesis has proceeded past the nascent leading strand. Black strands, parental DNA; red strands, nascent leading-strand DNA or invading-strand DNA; blue strands, nascent lagging-strand DNA; green strand, RNA of DNA-RNA hybrid. The arrowheads at the end of the nascent DNA represent the 3'-OH termini.
Figure 3: A Template for Reconstitution of Replication Restart at a Stalled Replication Fork--The template is blunt-ended on one side, contains 6.9 kb of duplex DNA, and has 38 nucleotide noncomplementary arms on the other side. Leading-strand oligonucleotides of different lengths can be annealed to the fork junction of the template. After replication, both the nascent leading- and lagging-strand DNA becomes labeled. Products are analyzed by alkaline agarose gel electrophoresis, which denatures double-stranded DNA. A representative DNA replication reaction is shown. The nascent leading strand migrates as a distinct band near the top of the gel whereas the heterogeneous population of nascent lagging-strand products migrates as a smear near the bottom of the gel.
Figure 4: Leading-Strand Gaps Differentially Affect Replication Restart--Protein requirements for PriA-dependent (A) and PriC-dependent (B) replication restart. The indicated proteins were omitted from the reaction mixtures. Either the full-length leading-strand oligonucleotide was annealed to the forked, linear template, or the leading-strand was shortened from the 3'-end to create gaps of the indicated sizes. Total DNA synthesis is indicated relative to the appropriate complete reaction in each case.
Figure 5--The leading strand can be reprimed during replication restart. (A) A stalled fork in which lagging-strand synthesis has progressed past the nascent leading strand is an excellent substrate for PriC-dependent restart. Arrowheads indicate the direction of 5'→3' DNA synthesis. DnaB is represented by the grey ovals. (B) A complete PriC restart reaction using the linear fork template with no oligos as substrate. Protein components are omitted as indicated. DNA synthesis is measured relative to the complete reaction.
Figure 6: Replication Fork Reactivation from Leading-Strand-Specific Lesions that Involves Nascent Leading-Strand Reinitiation--(A) At a replication fork where polymerase uncoupling has occurred because of a blockage in the leading-strand template, it is possible that DnaB can continue to unwind the template downstream of the lesion. The lagging-strand polymerase might also still be present. Under these circumstances, DnaG primase-directed re-priming of the nascent leading strand could result in re-establishment of the replisome with a resulting gap left behind in the nascent leading strand. (B) On the other hand, if the fork stalls completely and the replisome components dissociate, unwinding of nascent lagging-strand DNA by a 3'→5' helicase, such as PriA or Rep, provides sufficient single-stranded DNA for the loading of DnaB by the PriC-directed system. Through a protein-protein interaction between DnaB and DnaG, a RNA primer is synthesized on the leading-strand template, allowing reinitiation of the nascent leading strand. The fork progresses past the blocking lesion, leaving a single-stranded gap on the opposite strand. (C) For models that involve nascent-strand regression, the nascent leading- and lagging-strand DNA anneals together, and the fork reverses to form a four-way Holliday junction. The RuvABC branch migration/Holliday junction endonuclease can resolve the structure, allowing the recombination proteins to process the double-strand end and catalyse the formation of a D-loop. In the template-switching mode, the nascent leading strand is extended using the nascent lagging strand as template. By resetting the fork, the lesion is effectively bypassed. Alternatively, the lesion could be excised and the four-way junction reset to a fork by an exonuclease. All these processes would generate a structure that is recognized by PriA for restart. Black strands, parental DNA; red strands, nascent leading-strand DNA; blue strands, nascent lagging-strand DNA. The arrowheads at the end of the nascent DNA represent the 3'-OH termini.
Figure 7: Construction of a Linear DNA Template Containing Template Damage at a Specific Site--An oligonucleotide carrying either an abasic site or a cyclopyrimidine dimer is used to prime replication of the complementary strand of an M13 viral DNA. The strand is sealed using DNA ligase and the covalently-closed, circular DNA is isolated by ethidium bromide buoyant density gradient centrifugation. Using different combinations of restriction enzymes, the forked end of the template can be joined to linearized DNA in such a manner that either the nascent leading- or nascent lagging-strand will encounter the template damage.
Figure 8: Replisome Stalling and Restart on a DNA Template Carrying a Cyclopyrimidine Dimer in the Leading-Strand Template--Replisomes formed at the forked end of the linear template are capable of replicating the entire template in the absence of DNA damage, generating a full-length leading strand and lagging-strand Okazaki fragments. When the cyclopyrimidine dimer is present in the leading-strand template, the replisome stalls, generating a shorter leading strand product. In the system shown, replication restart is evident by the presence of a shorter leading-strand fragment corresponding to the distance between the DNA damage site and the end of the template.
Orderly progression of the replication fork is essential for the timely and accurate duplication of our genetic material. Although the bacterial replisome is highly processive, capable of synthesizing megabase-long stretches of nascent duplex DNA without dissociating from the template, it is subject to many obstacles that can arrest replication forks. These obstacles can be chemical damage to the template bases (such as the formation of bulky adducts, abasic sites, or intra-strand thymidine dimers as a result of UV irradiation), nicks in one of the template strands, or frank double-strand breaks in the template. It is also the case that frozen protein-DNA complexes and trains of slow-moving RNA polymerases can block replication fork progression. In bacteria, the arrested replication forks must be restarted rapidly or asymmetric chromosome segregation will occur resulting in the production of chromosome-less, anucleate daughter cells. Repair of the DNA lesion may proceed replication restart or, as we have recently shown, the replisome may simply jump the damage and continue downstream. We have elaborated the biochemical pathways required for replication reactivation at several different DNA structures that can form when replication fork progression is arrested (Figure 1).
Stalled replication forks take two general forms, either the nascent leading strand is ahead of the nascent lagging strand or vice-versa (Figure 2). To study replication restart on stalled replication forks we devised a novel linear template that allows us to vary the structure of the forked end where the replisome will be loaded (Figure 3). Using this template, we have shown that there are two distinct biochemical pathways optimized for using these different substrates (Figure 4). These replication systems utilize the chromosomal replicase, the DNA polymerase III holoenzyme (Pol III HE), itself a 10-subunit enzyme, the single-stranded DNA-binding protein (SSB), and different combinations of the primosomal proteins that both recognize the stalled replication fork structures and re-load the replisome [PriA, PriB, PriC, DnaT, DnaC, DnaB (the replication fork DNA helicase), and DnaG (the primase)]. Our biochemical data demonstrated that the PriA system (composed of PriA, PriB, DnaT, DnaB, DnaC, and DnaG) prefers stalled replication fork structures where there is no gap in the nascent leading strand, whereas the PriC system (PriC, DnaB, DnaC, and DnaG) prefers stalled fork structures where there is a gap in the nascent leading strand. Because the PriA system also restarts replication at recombinant joint molecules that are generated after the formation of double-strand breaks in the template (Figure 2), these two replication restart systems are sufficient to restart any stalled replication fork in the cell. These biochemical observations agree with those of our colleague, Steve Sandler, who studies the genetics of replication restart.
Our studies on the biochemical pathways of replication restart suggested to us that DnaG could prime leading-strand synthesis de novo (see Figure 4 where, with the PriC system, even though a nascent leading strand is present at the fork, leading-strand synthesis appears to be dependent on the presence of the primase). This was a heretical concept. It had been accepted wisdom for over 40 years that leading-strand synthesis could only be primed at origins of replication. However, as shown in Fig. 5, both leading- and lagging-strand synthesis can be primed by DnaG de novo using the PriC system (and, for that matter, also the PriA system). This observation has led us to propose that replisomes that stall at a lesion in the leading-strand template can simply re-initiate replication downstream, leaving the template damage and a gap in the nascent leading strand behind (Figure 6). The damage can be repaired subsequently and the gap repaired by daughter-strand gap repair.
To be able to study these processes we have developed a new method for generating our linear template with a thymidine dimer in a specific location (Figure 7). Using these templates carrying DNA damage we can observe directly replication fork stalling and restart (Figure 8).
Among the questions that we are interested in answering are: What is the fate of the replication proteins when a replisome stalls? What is the structure of these stalled replication forks? What is the role of replication fork regression in restart? What are the requirements for replication restart? Do restart requirements differ depending on the type of DNA template damage? How do replication restart and lesion bypass cooperate? Is there a specific mechanism that displaces the Pol III HE from the DNA? And, can the same replisome jump template damage and restart downstream?