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

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  • Haohui Duan, University of Massachusetts Boston
  • ,
  • Sarah Mansour, University of Massachusetts Boston
  • ,
  • Rachel Reed, Dana-Farber Cancer Institute
  • ,
  • Margaret K. Gillis, University of Massachusetts Boston
  • ,
  • Benjamin Parent, Dana-Farber Cancer Institute
  • ,
  • Ben Liu, Dana-Farber Cancer Institute
  • ,
  • Zsofia Sztupinszki, Danish Cancer Society
  • ,
  • Nicolai Birkbak
  • Zoltan Szallasi, Danish Cancer Society, Harvard University
  • ,
  • Andrew E.H. Elia, Harvard University
  • ,
  • Judy E. Garber, Dana-Farber Cancer Institute
  • ,
  • Shailja Pathania, University of Massachusetts Boston

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.

Original languageEnglish
Article numbere201908192
JournalThe Journal of Cell Biology
Number of pages27
Publication statusPublished - 2020

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