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

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.

MonitoringS. pombegenome stress by visualizing end-binding protein Ku

“…The most dramatic increase in fraction of cells with foci was observed with MMS at 173 0.9mM, an alkylating agent that results in error-free and error prone base excision repair during 174 S phase, and thus leading to trans lesion synthesis. This induction in MMS is consistent with 175 prior observations suggesting that Ku is recruited to regressed or broken replication forks in 176 order to stabilize the free end (Langerak, P., et al 2011;Teixeira-Silva, A., et al 2017). This 177 suggests that even in MRN + cells, there are situations where Ku remains associated with sites of 178 genome stress.…”

“…2018). and is essential to drive resection (Wang, Q., et al 2014;153 Shibata, A., et al 2018;Langerak, P., et al 2011;Teixeira-Silva, A., et al 2017). Our Mre11-154 mCherry construct showed a diffuse pan-nuclear signal in untreated cells.…”

“…In 163 unperturbed cells, there is a diffuse pan-nuclear localization. Association of Ku with DNA ends 164 has been investigated using ChIP methods; in wild type cells, it is not enriched unless the MRN 165 complex is missing (Langerak, P., et al 2011;Teixeira-Silva, A., et al 2017). Visualization of 166 Pku70 in live fission yeast cells has not previously been performed.…”

“…Ku is associated with the non-homologous end-joining (NHEJ) mechanism of 21 DNA double strand break (DSB) repair (Mahaney, B. L., et al 2009) and protects telomeres 22 (Baumann, P., & Cech, T. R., 2000;Ferreira, M. G., & Cooper, J. P. 2001). Additionally, it 23 recognizes "one-sided" double strand breaks and ends associated with regressed replication forks 24 (Teixeira-Silva, A., et al 2017;Foster, S. S., et al 2011;Langerak, P., et al 2011). 25 Ku binding to DNA ends inhibits the resection and accumulation of single strand DNA 26 that otherwise drives homologous recombination (Shibata, A., et al 2018).…”

“…To some degree, Ku and MRN act as mutual antagonists; Ku 35 inhibits short-range resection driven by MRN, and MRN removes Ku to facilitate homologous 36 recombination (HR) over NHEJ; and to prevent inappropriate repair of single-end breaks 37 (Langerak, P., et al 2011;Shao, Z., et al 2012;Myler, L. R., et al 2017;Shibata, A., et al 38 2018). Interestingly, loss of Ku partly suppresses the sensitivity to DNA damage and replication 39 blocking toxins associated with mutation of MRN (Tomita, K., et al 2003;Williams, R. S., et al 40 2008;Limbo, O., et al 2007;Langerak, P., et al 2011;Teixeira-Silva, A., et al 2017), which can 41 lead to excessive Exo1 driven resection but impaired RPA recruitment (Teixeira-Silva, A., et al 42 2017). 43 In this report, we describe the development of a new fluorescent marker for fission yeast, 44 the Pku70 subunit of the Ku protein complex that recognizes DNA ends.…”