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

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...

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Bibliographic Details
Published in:The Journal of cell biology 2020-06, Vol.219 (6), p.1
Main Authors: Duan, Haohui, Mansour, Sarah, Reed, Rachel, Gillis, Margaret K, Parent, Benjamin, Liu, Ben, Sztupinszki, Zsofia, Birkbak, Nicolai, Szallasi, Zoltan, Elia, Andrew E H, Garber, Judy E, Pathania, Shailja
Format: Article
Language:English
Subjects:
BRCA1 protein
BRCA1 Protein - deficiency
BRCA1 Protein - genetics
BRCA1 Protein - metabolism
BRCA2 protein
BRCA2 Protein - deficiency
BRCA2 Protein - genetics
BRCA2 Protein - metabolism
Breast cancer
Cancer
Cell Cycle
Cell Line, Tumor
Cell Survival - genetics
Collapse
Degradation
DNA biology
DNA Damage - drug effects
DNA Damage - genetics
DNA Helicases - genetics
DNA Helicases - metabolism
DNA Repair - genetics
DNA Replication - drug effects
DNA Replication - genetics
Humans
Hydroxyurea - pharmacology
MRE11 Homologue Protein - deficiency
MRE11 Homologue Protein - genetics
MRE11 Homologue Protein - metabolism
MRE11 protein
Mutation
Phosphorylation
Protein A
Rad51 Recombinase - metabolism
Repair
Replication
Replication forks
Replication protein A
Replication Protein A - metabolism
RNA, Small Interfering
Sensitivity
Stability
Ubiquitin-protein ligase
Ubiquitin-Protein Ligases - genetics
Ubiquitin-Protein Ligases - metabolism
Ubiquitination - genetics
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Summary: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.
ISSN:0021-9525
1540-8140
DOI:10.1083/jcb.201908192