Fork restart

Replication have to restart DNA synthesis after resolving replication stress. Many mechanisms are involved in replication fork restart. Among these mechanisms, firing of dormant origins due to a recruitment of an excess of the helicase MCM2-7 during late mitosis and early G1-phase. It has been shown in Xenopus laevis that only a fraction of these origins are fired during a normal S-phase due to checkpoint activity (Woodward et al., 2006). In the presence of replicative stress more origins are fired in order to rescue replication forks.

Consistent with this, partial knock-down of MCMs proteins in C. elegans (Woodward et al., 2006) or human cells (Ge et al., 2007), has no effect on normal replication but induces lethality and suppresses the use of dormant origins upon replication stress.

A mean to restart the fork is repriming behind the lesion, leaving ssDNA gaps that will be repaired during post-replication repair on both leading and lagging strands using translesion synthesis and recombination (see above) (Elvers et al., 2011; Lopes et al., 2006). The third major mechanism to restart DNA replication is fork regression and reversal. During this pathway, the newly synthesized leading- and lagging strands are unwound. Then, the parental strands anneal back after the block in order to establish dsDNA around the obstacle and make the lesion exposed to repair factors. At this point, the newly synthesized unwound strands anneal and form a reversed fork structure, called a "chicken foot structure" where typical replication fork (three-way junction) is converted into a four-way junction (Figure 4. 3) (Neelsen and Lopes, 2015). The formation of these structures was observed by extensive electron microscopic analyses coupled with 2D gel electrophoresis (Berti et al., 2013; Cotta-Ramusino et al., 2005; Follonier et al., 2013; Neelsen et al., 2013). Several proteins are involved in fork reversal, PARP is one of those (Ray Chaudhuri et al.). Defects in these factors, induce replication problems, genomic instability, and difficulties to resolve fork blocking, confirming their physiological role for fork restart (Atkinson and McGlynn, 2009).

Fork reversal is a complicated mechanism, not yet completely understood. Recent work has begun to elucidate some of the many cellular factors required for fork remodelling in vivo.

When replication forks are uncoupled, ssDNA stretches accumulate and the recombinase RAD51 partially replaces RPA and converts these forks into reversed forks, especially following topoisomerase inhibition, nucleotide depletion, and in the presence of ICLs (Zellweger et al., 2015). Therefore, RAD51 loading at the extended ssDNA regions can prime

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fork reversal, promoting the re-annealing of parental strands (Figure 4. 3 a). Interestingly, in low nucleotide availability condition, replication fork reversal partially depends on the human F-Box DNA Helicase protein 1 (FBH1) (Fugger et al., 2015). FBH1 presumably initiates the unwinding of the lagging strand (Masuda-Ozawa et al., 2013) (Figure 4. 3 a). In addition to RAD51 and FBH1, several other factors belonging to the Fanconi anaemia or homologous recombination pathways such as the breast cancer susceptibility proteins BRCA1 and BRCA2, the RAD51 paralogues, and FANCD2 were shown to participate in different steps of replication fork remodelling (Kim and D'Andrea, 2012) (Figure 4. 3 a).

Moreover, the SMARCAL1/HARP protein is a DNA translocase that can re-anneal ssDNA bubbles coated by RPA and is probably involved in the parental strands re-annealing during regression (Blastyak et al., 2007; Yusufzai and Kadonaga, 2008). SLX4 nuclease in complex with SLX1 was recently shown to participate to the cleavage of replication forks regressed by SMARCAL1/HARP (Couch et al., 2013). This complex can process fork structures and Holliday junctions (Fekairi et al., 2009; Svendsen et al., 2009). The FancM helicase is also capable of unwinding both fork structures and double Holliday-structures and catalyses fork regression in vitro (Gari et al., 2008).

Once reversed forks are formed, ssDNA regions on the regressed arm are expanded by nucleolytic degradation and recruit more RAD51, promoting invasion of the re-annealed homologue template strands and thus fork restarts in recombination-mediated manner (Figure 3.10a). Other mechanisms are involved in the restart or reversed forks. For instance, the RECQ1 ATP-dependent DNA helicase is able to bind reversed forks and restart them by branch migration (Figure 4. 3 b). In this pathway, RECQ1 is only activated when PARP is inactivated by replication stress and DNA repair signalling (Berti et al., 2013; Zellweger et al., 2015).

Another pathway is mediated by the combined action of nucleolytic degradation and unwinding of the regressed arm using the ATP-dependent helicase–nuclease (DNA2) and the Werner syndrome helicase. Then, the recombination-dependent restart occurs (Thangavel et al., 2015)Another pathway is mediated by the combined action of nucleolytic degradation and unwinding of the regressed arm using the ATP-dependent helicase–nuclease (DNA2) and the Werner syndrome helicase. Then, the recombination-dependent restart occurs (Thangavel et al., 2015).

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Figure 4. 3Fork reversal as a mechanism for fork restart (Neelsen and Lopes, 2015)

(A) The involvement of homologous recombination factors in replication fork remodelling. Upon replication stress and fork uncoupling, RPA coating ssDNA is partially replaced by RAD51. Controlled resection of newly synthesized strands assists RAD51 loading and is mediated probably by the Fanconi anaemia (FA) and homologous recombination (HR) factors. RAD51mediates homology search and strand invasion promoting the reannealing of parental strands, and priming fork reversal, which is assisted by F̻box-containing DNA helicase 1 (FBH1)-mediated displacement of the growing lagging strand. Once reversed forks are formed, ssDNA regions on the regressed arm may also recruit RAD51, and promote homology-driven invasion of the reannealed template strands and thus result in recombination-mediated fork restart. (B) Restart mechanisms of reversed forks. The DNA helicase RECQ1 binds reversed forks and promotes branch migration to restart the fork. PARP transiently inhibit RECQ1 which is only reactivated (RECQ1*) when DNA repair and replication stress release allow local PARP inactivation. RECQ1 binding inhibits another pathway involved in reversed fork restart, consisting in the unwinding and the nucleolytic degradation of the regressed arm by the nuclease activities of WRN helicase and DNA2 helicase/nuclease. The resected regressed arm may promote the recruitment of branch migration factors or homologous recombination-dependent restart.

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