Perturbing cohesin dynamics drives MRE11 nuclease-dependent replication fork slowing

Abstract

Pds5 is required for sister chromatid cohesion, and somewhat paradoxically, to remove cohesin from chromosomes. We found that Pds5 plays a critical role during DNA replication that is distinct from its previously known functions. Loss of Pds5 hinders replication fork progression in unperturbed human and mouse cells. Inhibition of MRE11 nuclease activity restores fork progression, suggesting that Pds5 protects forks from MRE11-activity. Loss of Pds5 also leads to double-strand breaks, which are again reduced by MRE11 inhibition. The replication function of Pds5 is independent of its previously reported interaction with BRCA2. Unlike Pds5, BRCA2 protects forks from nucleolytic degradation only in the presence of genotoxic stress. Moreover, our iPOND analysis shows that the loading of Pds5 and other cohesion factors on replication forks is not affected by the BRCA2 status. Pds5 role in DNA replication is shared by the other cohesin-removal factor Wapl, but not by the cohesin complex component Rad21. Interestingly, depletion of Rad21 in a Pds5-deficient background rescues the phenotype observed upon Pds5 depletion alone. These findings support a model where loss of either component of the cohesin releasin complex perturbs cohesin dynamics on replication forks, hindering fork progression and promoting MRE11-dependent fork slowing.

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

“…Our results are consistent with those recently published by Carvajal-Maldonado et al (52), who first showed that depletion of PDS5 proteins results in replication fork progression defects that are rescued by MRE11 inhibition. However, they differ in two important aspects.…”

“…Moreover, we show that both PDS5A and PDS5B interact with fork protection pathway components. It is conceivable that the ratio of cohesin to PDS5A/B is slightly different in HeLa cells (used in our study) and U2OS cells used by Carvajal-Maldonado et al (52). Alternatively, additional factors may account for the differences in our results.…”

“…In con-trast, Carvajal-Maldonado et al found similar defects after depleting only PDS5A, PDS5B, or WAPL. Regarding the connection between PDS5 and BRCA2, Carvajal-Maldonado et al (52) argue that PDS5 binding to chromatin is independent of BRCA2, the latter being only required in replication stress conditions. Although this is true, it is important to note that the presence of unreleased cohesin in PDS5-deficient cells generates stalled forks but depletion of BRCA2 does not.…”

PCNA antagonizes cohesin-dependent roles in genomic stability

“…Our findings document that PCNA OE is sufficient to produce growth defects in mcd1-1 mutant cells, and that this effect is greatly exacerbated by challenging mutant cells with MMS or HU. This intersection between PCNA and cohesin is consistent with prior studies that document that 1) highly elevated levels of PCNA result in genomic instability [63], 2) cohesin are both recruited to stalled replication forks and promote fork restart [77][78][79]89], and 3) DNA replication fork protection complexes and cohesin pathways are intimately linked [24,63,77,80,108]. During the final stages of this study, independent analyses revealed that elevated levels of chromatin-bound PCNA results in hyperrecruitment of mismatch repair factors [109].…”

“…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.…”