Replication Fork Reversal during DNA Interstrand Crosslink Repair Requires CMG Unloading

DNA 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)...

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Bibliographic Details
Published in:Cell reports (Cambridge) 2018-06, Vol.23 (12), p.3419-3428
Main Authors: Amunugama, Ravindra, Willcox, Smaranda, Wu, R. Alex, Abdullah, Ummi B., El-Sagheer, Afaf H., Brown, Tom, McHugh, Peter J., Griffith, Jack D., Walter, Johannes C.
Format: Article
Language:English
Subjects:
Animals
Cell Extracts
CMG
Cross-Linking Reagents - metabolism
DNA - metabolism
DNA Helicases - metabolism
DNA interstrand crosslink repair
DNA Repair
DNA Replication
DNA-Binding Proteins - metabolism
Fanconi anemia
ICL
Ovum - metabolism
replication fork reversal
Xenopus laevis - metabolism
Xenopus Proteins - metabolism
XPF
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Summary:DNA 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. [Display omitted] •Replication forks undergo reversal during repair of a defined DNA interstrand crosslink•Fork reversal requires replicative CMG helicase unloading•DNA incisions occur in the context of a single fork DNA interstrand crosslinks (ICLs) are extremely cytotoxic lesions that are mainly repaired in a replication-coupled manner. Using a cell-free system, Amunugama et al. report that, during ICL repair, replication forks undergo reversal. Fork reversal requires replicative CMG helicase unloading.
ISSN:2211-1247
2211-1247
DOI:10.1016/j.celrep.2018.05.061