ATR Promotes the Replication Traverse of DNA Interstrand Crosslinks

Abstract

DNA instability disorders are associated with cancer, neurodegeneration, and premature aging. The genome integrity is most vulnerable during S phase during which the replication apparatus encounters many impediments. Among the most severe are interstrand crosslinks (ICLs) which are absolute blocks to helicases, and have always been regarded as absolute blocks to replication. Cells from patients with the genome instability disorder Fanconi Anemia (FA) are highly sensitive to crosslinking agents. We have developed a novel single molecule strategy, based on DNA fibers, to visualize collisions of replication forks with ICLs. Remarkably, we found that DNA synthesis can resume past an ICL, leaving behind still crosslinked parental strands, a process that requires only a few minutes. Traverse of ICLs requires the activity of the Fanconi Anemia translocase FANCM. Furthermore, while the FA “core” complex proteins, which monoubiquitinate the FANCD2 protein, were not required, FANCD2 was. These results indicate that non ubiquitinated FANCD2 is involved in replication traverse of the ICLs. The most profound suppression of replication restart patterns was observed in cells deficient in the replication stress kinase ATR, among whose substrates are FANCM and FANCD2. ATR is required for the recruitment of FANCM to ICLs. Cells expressing a mutant form of FANCM that cannot be phosphorylated by ATR show a marked decline in the frequency of replication traverse events at ICLs. This research was supported entirely by the Intramural Research Program of the NIH, National Institute on Aging.