Wednesday, February 5, 2025
2/5/2025
ABSTRACT DNA double strand breaks (DSBs) are induced by environmental and chemotherapeutic agents, and during encounters of the DNA replication machinery with DNA damage. The two major, mechanistically distinct DSB repair pathways are non-homologous DNA end joining (NHEJ) and homology-directed repair (HDR). NHEJ is efficient but error-prone, while HDR is inherently accurate and represents the preferred repair tool for DNA replication-associated DSBs. HDR commences with the resection of the 5’-terminated strand at break ends to generate a DNA tail that serves as the template for assembly of the RAD51 recombinase filament. DSB repair pathway choice is linked to cell cycle progression and is determined by whether or not a DSB undergoes extensive resection. Long-range resection is principally mediated by the 5’-3’ exonuclease EXO1 or the BLM helicase-DNA2 endonuclease. The chromatin reader 53BP1 nucleates the formation of a higher order ensemble that harbors the RIF1 protein and the hetero-tetrameric Shieldin complex at DSB ends to block end resection in the G1 phase of the cell cycle. The restrictive action of the 53BP1 axis is alleviated by BRCA1- BARD1 in S and G2 phases via mechanisms that are poorly understood. Thus, BRCA1-deficient tumors, on account of their HDR-deficiency, are particularly vulnerable to PARP inhibitors (PARPi) due to synthetic lethality. However, dysfunction in the 53BP1 axis leads to HDR restoration and PARPi resistance. Importantly, we now have compelling evidence that RIF1 and Shieldin strongly restrict the activity of the DNA end resection enzymes. To elucidate the underpinnings of the DNA end resection restriction circuitry, we will employ a combinatorial approach encompassing reconstitution biochemistry and cell biology to: (1) Delineate how RIF1 and Shieldin interfere with the activity of the 5’-3’ exonuclease EXO1 and of the helicase-endonuclease complex BLM-DNA2 in DNA end resection and (2) Interrogate BRCA1-BARD1 for its ability to overcome the restriction of DNA end resection imposed by RIF1 and Shieldin. Our studies will elucidate the intricate regulatory networks that control DNA end resection onset and efficiency. Our endeavors will not only illuminate the mechanistic principles of DSB repair pathway choice, but will also exert a major impact in our understanding of how failure to properly process DSBs lead to neoplastic cell transformation and cancer, and will provide actionable information to help guide the development of targeted cancer therapies to treat BRCA- deficient cancers and circumvent drug resistance.
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