• E3 ligase RFWD3 is a novel modulator of stalled fork stability in BRCA2-deficient cells.

E3 ligase RFWD3 is a novel modulator of stalled fork stability in BRCA2-deficient cells.

The Journal of cell biology (2020-05-12)
Haohui Duan, Sarah Mansour, Rachel Reed, Margaret K Gillis, Benjamin Parent, Ben Liu, Zsofia Sztupinszki, Nicolai Birkbak, Zoltan Szallasi, Andrew E H Elia, Judy E Garber, Shailja Pathania

BRCA1/2 help maintain genomic integrity by stabilizing stalled forks. Here, we identify the E3 ligase RFWD3 as an essential modulator of stalled fork stability in BRCA2-deficient cells and show that codepletion of RFWD3 rescues fork degradation, collapse, and cell sensitivity upon replication stress. Stalled forks in BRCA2-deficient cells accumulate phosphorylated and ubiquitinated replication protein A (ubq-pRPA), the latter of which is mediated by RFWD3. Generation of this intermediate requires SMARCAL1, suggesting that it depends on stalled fork reversal. We show that in BRCA2-deficient cells, rescuing fork degradation might not be sufficient to ensure fork repair. Depleting MRE11 in BRCA2-deficient cells does block fork degradation, but it does not prevent fork collapse and cell sensitivity in the presence of replication stress. No such ubq-pRPA intermediate is formed in BRCA1-deficient cells, and our results suggest that BRCA1 may function upstream of BRCA2 in the stalled fork repair pathway. Collectively, our data uncover a novel mechanism by which RFWD3 destabilizes forks in BRCA2-deficient cells.


抗磷酸组蛋白H2A.X(Ser139)抗体,克隆JBW301, clone JBW301, Upstate®, from mouse
Isopore滤膜, Isopore, filter diam. 47 mm, hydrophilic
抗-BRCA1抗体, serum, Upstate®
Anti-BRCA1 (Ab-4) Mouse mAb (SD118), liquid, clone SD118, Calbiochem®
Rabbit anti-GRLF1/p190RhoGAP Antibody, Affinity Purified, Powered by Bethyl Laboratories, Inc.