Rescuing stalled or damaged replication forks

Abstract

In recent years, an increasing number of studies have shown that prokaryotes and eukaryotes are armed with sophisticated mechanisms to restart stalled or collapsed replication forks. Although these processes are better understood in bacteria, major breakthroughs have also been made to explain how fork restart mechanisms operate in eukaryotic cells. In particular, repriming on the leading strand and fork regression are now established as critical for the maintenance and recovery of stalled forks in both systems. Despite the lack of conservation between the factors involved, these mechanisms are strikingly similar in eukaryotes and prokaryotes. However, they differ in that fork restart occurs in the context of chromatin in eukaryotes and is controlled by multiple regulatory pathways.

Figure 1.

Bypass of leading-strand template damage by leading-strand repriming in E. coli. Following a collision with a leading-strand lesion, template unwinding and lagging-strand synthesis are believed to continue beyond the site of damage. (A) Repriming of the leading strand can occur downstream from the damage, which enables replication to continue without the replisome dissociating from the DNA (Yeeles and Marians 2011). (B) Should the replisome dissociate following the collision, the replication restart protein PriC can reload DnaB, which enables replication to be reinitiated by priming the leading strand downstream from the damage (Heller and Marians 2006b).

Figure 2.

Bypass of leading-strand template damage in eukaryotes. As in bacteria, template unwinding and lagging-strand synthesis are believed to continue beyond the site of damage, resulting in the formation of an ssDNA gap. (A) Fork-associated lesion bypass allows the restart of leading-strand synthesis upon PCNA modification and the transient recruitment of a mutagenic TLS polymerase. (B) Repriming of the leading strand downstream from the lesion leaves an ssDNA gap. (C) This gap can be repaired postreplicatively by TLS polymerases. (D) Error-free lesion bypass can also be performed through a recombination-mediated mechanism called a template switch, which uses the newly synthesized sister chromatid as a template for primer elongation. Note that template switching can also occur after fork regression, as illustrated in Figure 4B. (E) Incomplete nucleotide excision repair of the DNA lesion or cleavage of the fork by endonucleases may also lead to the formation of a one-ended DSB, which can be repaired by an HR-related process called break-induced replication.

Figure 3.

Models for replication fork regression at E. coli replication forks stalled by leading-strand template damage. The helicase RecG binds with high affinity to forks containing a leading-strand gap and unwinds the structure to generate a Holliday junction. A second pathway of fork regression involves RecA binding to the single-stranded region of the fork to drive fork regression and potentially form a Holliday junction. (A) Regression of the fork places the original lesion in a region of double-stranded DNA, enabling it to be repaired by NER. Exonucleolytic degradation of the lagging-strand extension resets the fork to enable replisome loading. (B) The nascent leading strand that is displaced by the regression reaction can be extended using the lagging strand as template. Rewinding of the Holliday junction, possibly by RecG, will place the nascent 3′ end of the leading strand beyond the site of damage to enable replication restart to occur without a need to reprime the leading strand.

Figure 4.

Models for replication fork regression at eukaryotic forks stalled by leading-strand template damage. Several DNA helicases have been shown to promote fork regression in vitro in yeast and vertebrates, including Rad5/HLTF and FANCM/Fml1. In vertebrates, fork regression in vivo also depends on poly(ADP-ribose) polymerase (PARP) (Ray Chaudhuri et al. 2012). Resumption of DNA replication after repair of the lesion (A) or template switching (B) is mediated by nucleolytic degradation of branched structures or reverse branch migration, as described for bacteria. (C) Fork restart can also occur after invasion of the duplex ahead of the lesion by the 3′ overhang of the forked structure. The resulting Holliday junctions are resolved by resolvases or dissolved by the combined action of the RecQ helicases BLM/Sgs1 and the type I topoisomerase Top3, whose function is conserved from yeasts to human.