Replication Fork Reversal during DNA Interstrand Crosslink Repair Requires CMG Unloading

Abstract

SUMMARYDNA interstrand crosslinks (ICLs) are extremely cytotoxic, but the mechanism of their repair remains incompletely understood. Using Xenopus egg extracts, we previously showed that repair of a cisplatin ICL is triggered when two replication forks converge on the lesion. After CDC45/MCM2-7/GINS (CMG) ubiquitylation and unloading by the p97 segregase, FANCI-FANCD2 promotes DNA incisions by XPF-ERCC1, leading to ICL unhooking. Here, we report that, during this cell-free ICL repair reaction, one of the two converged forks undergoes reversal. Fork reversal fails when CMG unloading is inhibited, but it does not require FANCI-FANCD2. After one fork has undergone reversal, the opposing fork that still abuts the ICL undergoes incisions. Our data show that replication fork reversal at an ICL requires replisome disassembly. We present a revised model of ICL repair that involves a reversed fork intermediate.

Replisome preservation by a single-stranded DNA gate in the CMG helicase

“…We refer to this mechanism as “replisome preservation”: while CMGM itself can nucleate a replisome by recruiting soluble components, it is also possible that other CMG-associated factors, or even the entire replisome, stay with CMG during these transitions. When two opposing forks converge upon replication termination or during interstrand crosslink repair, CMG is demonstrated to be targeted by the ubiquitin ligase and segregase for unloading from DNA (Amunugama et al, 2018; Maric et al, 2014). But whether it is converging CMGs, CMG binding to dsDNA, or some other signal that triggers CMG ubiquitylation remains to be established.…”

Fanconi anemia pathway as a prospective target for cancer intervention

“…ICLs that occur outside of S phase are sensed and repaired by the NER pathway [14]. The FA pathwaymediated ICL repair occurs primarily in S phase and starts with the formation of an X shaped DNA structure that occurs upon convergence of two head-on replication forks surrounding the ICL site [24,25] (Fig. 1a).…”

Cdc48 regulates intranuclear quality control sequestration of the Hsh155 splicing factor

“…S1D) (16,17). In addition, Cdc48 regulates protein-DNA complexes, such as the removal of RNA polymerase from damaged DNA (15), the removal of replisomes during DNA replication termination (14), at protein-DNA crosslinks (21), and in the release of condensin complexes (22). Thus, Cdc48 is well-positioned to regulate protein sequestration pathways such as INQ following DNA damage detection.…”