PDS5 proteins are required for proper cohesin dynamics and participate in replication fork protection

Abstract

Edited by Patrick Sung Cohesin is a chromatin-bound complex that mediates sister chromatid cohesion and facilitates long-range interactions through DNA looping. How the transcription and replication machineries deal with the presence of cohesin on chromatin remains unclear. The dynamic association of cohesin with chromatin depends on WAPL cohesin release factor (WAPL) and on PDS5 cohesin-associated factor (PDS5), which exists in two versions in vertebrate cells, PDS5A and PDS5B. Using genetic deletion in mouse embryo fibroblasts and a combination of CRISPRmediated gene editing and RNAi-mediated gene silencing in human cells, here we analyzed the consequences of PDS5 depletion for DNA replication. We found that either PDS5A or PDS5B is sufficient for proper cohesin dynamics and that their simultaneous removal increases cohesin's residence time on chromatin and slows down DNA replication. A similar phenotype was observed in WAPL-depleted cells. Cohesin down-regulation restored normal replication fork rates in PDS5-deficient cells, suggesting that chromatin-bound cohesin hinders the advance of the replisome. We further show that PDS5 proteins are required to recruit WRN helicase-interacting protein 1 (WRNIP1), RAD51 recombinase (RAD51), and BRCA2 DNA repair associated (BRCA2) to stalled forks and that in their absence, nascent DNA strands at unprotected forks are degraded by MRE11 homolog double-strand break repair nuclease (MRE11). These findings indicate that PDS5 proteins participate in replication fork protection and also provide insights into how cohesin and its regulators contribute to the response to replication stress, a common feature of cancer cells. This work was supported by the Spanish Ministry of Economy and Competitiveness and FEDER Grants BFU2013-48481-R and BFU2016-79841-R (to A. L.) and BFU2016-80402-R (to J. M.) and by FPI "Severo Ochoa" fellowships (to C. M. and M. R.-T.). This work was also supported by funding from Boehringer Ingelheim Fonds (to M. R.-T.). The authors declare that they have no conflicts of interest with the contents of this article. Author's Choice-Final version open access under the terms of the Creative Commons CC-BY license. This article contains Figs. S1-S5 and Tables S1 and S2.

PDS 5 proteins regulate the length of axial elements and telomere integrity during male mouse meiosis

“…Likewise, PDS5 proteins in mouse could mediate RAD51 loading to early recombination nodules, as in vitro assays show that PDS5B interacts physically with RAD51 and stimulates RAD51‐mediated DNA strand invasion . In this regard, we have recently showed in human cells that PDS5A and PDS5B promote the recruitment of RAD51 and BRCA2 to stalled replication forks to protect them from excessive resection by MRE11 nuclease .…”

PCNA antagonizes cohesin-dependent roles in genomic stability

“…PCNA OE sensitizes mcd1-1 mutant cells to MMS and HU and also activates the Mec1/ATR intra-S phase checkpoint, suggesting that elevated PCNA levels may adversely impact replication fork progression. Consistent with this hypothesis, mutation of either ESCO2 (human homolog of yeast ECO1) or cohesin subunits result in slowed fork progression in mammalian cells [86][87][88][89], although a velocity reduction appears absent in analogous yeast mutant strains [2,3,10,77,80,[90][91][92]. It thus became important to test whether elevated levels of chromatinbound PCNA produces replication stress severe enough to delay S phase progression in mcd1-1 mutant cells.…”

“…restart and repair [77][78][79]88], 3) Eco1-dependent cohesion establishment is intimately coupled to PCNA and the DNA replication fork [10,15,16,[25][26][27][121][122][123], and 4) PCNA OE and elg1Δ both adversely impact cohesin mutant cell growth. Clearly, PCNA OE (at even very moderate levels) and elg1Δ adversely impact cohesin mutant cell growth, an effect exacerbated by genotoxic agents.…”

Roles of OB-Fold Proteins in Replication Stress

“…Recent studies show that the PDS5-wings apart-like protein homolog (WAPL) complex, a cohesin-associated factor that releases cohesin from chromosomes, is also involved in replication fork progression ( Carvajal-Maldonado et al, 2019 ; Morales et al, 2020 ). Cohesin binds to chromatin in a multi-subunit complex that mediates cohesion between sister chromatids, but its role in replication and transcription remains unclear.…”