Figure 1: Recombination-Dependent Replication and Direct Restart Mechanisms of Reactivating Forks Stalled by Leading-Strand-Specific LesionsWhen 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 ForkThe 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 RestartProtein 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 5The 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 SiteAn 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 TemplateReplisomes 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.