Mutations in the hereditary breast/ovarian cancer genes BRCA1/2 were shown to be synthetic lethal with poly(ADP-ribose) polymerase inhibitors (PARPi). This toxicity is assumed to derive from PARPi-induced DNA double strand breaks (DSBs) that necessitate BRCA function in homologous recombination (HR) and/or fork protection (FP). However, PARPi accelerates replication forks. While high-speed replication could cause DSBs, the finding that PARPi leads to single-stranded DNA (ssDNA) gaps/nicks suggests replication gaps could also or alone be the cause of synthetic lethality.
Here, we demonstrate that PARPi toxicity derives from replication gaps. Isogenic cells deficient in BRCA1 or the BRCA1-associated FANCJ, with common DNA repair defects in HR and FP, exhibit opposite responses to PARPi. Deficiency in FANCJ, a helicase also mutated in hereditary breast/ovarian cancer and Fanconi anemia, causes aberrant accumulation of fork remodeling factor HLTF and limits unrestrained DNA synthesis with ssDNA gaps. Thus, we predict replication gaps as a distinguishing factor and further uncouple HR, FP and fork speed from PARPi response. BRCA-deficient cells display excessive gaps that are diminished upon resistance, restored upon re-sensitization and when targeted augment synthetic lethality with PARPi. Furthermore, we define the source of gaps to defects in Okazaki fragment processing (OFP). Unchallenged BRCA1-deficient cells have elevated poly(ADP-ribose) and chromatin-associated PARP1 but aberrantly low XRCC1 indicating a defective backup OFP pathway. Remarkably, 53BP1 loss resuscitates OFP by restoring XRCC1-LIG3 that suppresses the sensitivity of BRCA1-deficient cells to drugs targeting OFP or generating gaps. Collectively, our study highlights unprotected lagging strand gaps as a determinant of synthetic lethality, providing a new paradigm and biomarker for PARPi toxicity.
| Identifer | oai:union.ndltd.org:umassmed.edu/oai:escholarship.umassmed.edu:gsbs_diss-2179 |
| Date | 03 January 2022 |
| Creators | Cong, Ke |
| Publisher | eScholarship@UMassChan |
| Source Sets | University of Massachusetts Medical School |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Morningside Graduate School of Biomedical Sciences Dissertations and Theses |
| Rights | Copyright is held by the author, with all rights reserved., select |
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