The end-joining factor Ku acts in the end-resection of double strand break-free arrested replication forks

Ana Teixeira-Silva,Sarah A E Lambert,Julien Hardy,Ismail Iraqui,Karine Fréon,Marina Charlotte Nocente,Anissia Ait Saada

doi:10.1038/s41467-017-02144-5

Abstract

Replication requires homologous recombination (HR) to stabilize and restart terminally arrested forks. HR-mediated fork processing requires single stranded DNA (ssDNA) gaps and not necessarily double strand breaks. We used genetic and molecular assays to investigate fork-resection and restart at dysfunctional, unbroken forks in Schizosaccharomyces pombe. Here, we report that fork-resection is a two-step process regulated by the non-homologous end joining factor Ku. An initial resection mediated by MRN-Ctp1 removes Ku from terminally arrested forks, generating ~110 bp sized gaps obligatory for subsequent Exo1-mediated long-range resection and replication restart. The mere lack of Ku impacts the processing of arrested forks, leading to an extensive resection, a reduced recruitment of RPA and Rad51 and a slower fork-restart process. We propose that terminally arrested forks undergo fork reversal, providing a single DNA end for Ku binding. We uncover a role for Ku in regulating end-resection of unbroken forks and in fine-tuning HR-mediated replication restart.

Highlights

Replication requires homologous recombination (HR) to stabilize and restart terminally arrested forks
DSBs are repaired by the non-homologous end joining (NHEJ) pathway which promotes the direct ligation of DNA ends with limited or no end-resection[18]
Upon Rtf[1] expression, > 90% of forks travelling in the main replication direction are blocked at the RTS1-replication fork barriers (RFBs) resulting in dysfunctional forks

Summary

Introduction

Replication requires homologous recombination (HR) to stabilize and restart terminally arrested forks. We report that fork-resection is a two-step process regulated by the non-homologous end joining factor Ku. An initial resection mediated by MRN-Ctp[1] removes Ku from terminally arrested forks, generating ~110 bp sized gaps obligatory for subsequent Exo1-mediated longrange resection and replication restart. A DNA nick directly converts an active fork into a broken fork, accompanied with a loss of some replisome components[7] Forks lacking their replication competence, and terminally arrested, are often referred to as collapsed forks, whether broken or not. Ku is involved in the repair of replication-born DSBs, where it limits end-resection[21,22,23,24] and yeast Ku acts as a backup to promote cell survival upon replication stress[25,26]

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Results

Conclusion