p97 Promotes a Conserved Mechanism of Helicase Unloading during DNA Cross-Link Repair

Abstract

Interstrand cross-links (ICLs) are extremely toxic DNA lesions that create an impassable roadblock to DNA replication. When a replication fork collides with an ICL, it triggers a damage response that promotes multiple DNA processing events required to excise the cross-link from chromatin and resolve the stalled replication fork. One of the first steps in this process involves displacement of the CMG replicative helicase (comprised of Cdc45, MCM2-7, and GINS), which obstructs the underlying crosslink. Here we report that the p97/Cdc48/VCP segregase plays a critical role in ICL repair by unloading the CMG complex from chromatin. Eviction of the stalled helicase involves K48-linked polyubiquitylation of MCM7, p97-mediated extraction of CMG, and a largely degradation-independent mechanism of MCM7 deubiquitylation. Our results show that ICL repair and replication termination both utilize a similar mechanism to displace the CMG complex from chromatin. However, unlike termination, repair-mediated helicase unloading involves the tumor suppressor protein BRCA1, which acts upstream of MCM7 ubiquitylation and p97 recruitment. Together, these findings indicate that p97 plays a conserved role in dismantling the CMG helicase complex during different cellular events, but that distinct regulatory signals ultimately control when and where unloading takes place. Interstrand cross-links (ICLs) are extremely toxic DNA lesions that covalently couple both strands of the DNA duplex. Replicating cells are particularly sensitive to ICLs, which disrupt the strand separation required for DNA replication and transcription (1). Repair of ICLs is initiated primarily during S phase when a replication fork collides with the cross-link (2-4). Fork stalling triggers a damage response that involves multiple DNA processing events that promote excision of the ICL from chromatin and resolution of the stalled replication fork (5). Defects in ICL repair are associated with delays in cell cycle progression, increased chromosomal breakage, and severe sensitivity to DNA cross-linking agents. The cellular symptoms of deficient or aberrant repair are thought to underlie the molecular basis of several cancer predisposition syndromes (6), including Fanconi anemia (FA) and hereditary breast and ovarian cancer.Fanconi anemia is a rare chromosomal instability disorder caused by mutations in 1 of at least 16 different genes (7). Four of the FA genes are classified as breast/ovarian cancer susceptibility (BRCA) genes (FANCD1/BRCA2, FANCJ/BRIP1, FANCN/ PALB2, FANCO/RAD51C) (8), with recent evidence also supporting classification of BRCA1 as a new FA subtype (FANCS) (9). Together, the FA/BRCA proteins and associated factors form a multifunctional network that plays a critical role in managing various forms of DNA damage and replication stress (10). Although both FA and BRCA proteins are required for ICL repair, each pathway also supports nonoverlapping functions (11, 12) that play different roles in how cells respond to treatment with DNA cross-linking agents.Xenop...

Recent discoveries in the molecular pathogenesis of the inherited bone marrow failure syndrome Fanconi anemia

“…Recent studies have shown that BRCA1/FANCS is necessary for the removal of the CMG replicative helicase during ICL repair. 76 And previous studies indicate that BRCA1 competes with 53BP1 early in the repair process to promote HR and restrict error-prone NHEJ. 77, 78 Therefore, it seems likely that BRCA1/FANCS may play several distinct roles during the process of ICL repair.…”

The CMG helicase bypasses DNA protein cross-links to facilitate their repair

“…To address whether CMG dissociates from or translocates past a stable DPC, we examined approach in the presence of an inhibitor of the p97 ATPase (p97i), which is required to extract CMG from chromatin during replication termination and ICL repair (Dewar et al, 2017;Fullbright et al, 2016;Maric et al, 2014;Moreno et al, 2014;Semlow et al, 2016). If CMG has to dissociate upon stalling at the DPC to enable approach ( Figure 1E), p97i should delay approach.…”

Ufd1-Npl4 Recruit Cdc48 for Disassembly of Ubiquitylated CMG Helicase at the End of Chromosome Replication

“…Ubiquitylated CMG is normally disassembled so quickly that it is undetectable in wild-type cells, but it can be stabilized by inactivation of Cdc48 (Maric et al., 2014), which is required to disrupt the ubiquitylated helicase into its component parts, namely Cdc45, Mcm2–7, and GINS. Work with extracts of Xenopus laevis eggs also demonstrated a role for p97/Cdc48 in release of ubiquitylated CMG from chromatin at the end of chromosome replication (Fullbright et al., 2016, Moreno et al., 2014, Semlow et al., 2016), indicating that the principal features of DNA replication termination have been conserved throughout the course of eukaryotic evolution.…”