Thursday, April 3, 2025
4/3/2025
DNA replication stress in cancer - Summary Single-stranded (ss) DNA gaps have emerged as key determinants of genome stability and PARP inhibitor (PARPi) sensitivity, particularly in the context of cancers with mutations in the breast cancer susceptibility genes BRCA1 and BRCA2. During this funding period, we unveiled a central role for PRIMPOL-repriming in generating ssDNA gaps in cells treated with platinum-based compounds and PARPi. This work was enabled by the unique combination of electron microscopy (EM) and single-molecule DNA fiber approaches available in our lab. Using these technologies, we provided the first molecular insight into the mechanisms that repair PRIMPOL-dependent gaps. We also showed that these mechanisms are impaired in BRCA1-deficient cells, explaining why gaps accumulate in these cells. However, we found that gap repair can be restored in BRCA1-deficient cells by inhibiting MRE11 nuclease activity, indicating that nuclease processing plays a central role in ssDNA gap repair. This renewal builds on new EM data showing that the ssDNA gaps in BRCA1-deficient cells treated with PARPi are significantly larger than the ssDNA gaps of BRCA1-proficient cells, and on new biochemical data showing that MRE11 efficiently resects ssDNA gaps in vitro. Based on these findings, we hypothesize that MRE11 plays a key role in processing ssDNA gaps, and that over-resection of the ssDNA gaps by MRE11 in BRCA1-deficient cells prevents gap repair. The first Aim will define the mechanism(s) by which ssDNA gaps are processed by MRE11, in concert with other long-resection nucleases. While processing of double-stranded breaks has been widely studied, the same molecular details are not available for ssDNA gaps. These studies will establish a new paradigm for the roles of nucleases in ssDNA gap resection and repair at DNA replication forks, as well as elucidate the mechanisms controlling nuclease activity and how these are deregulated in BRCA-deficient tumors. We discovered that inhibiting MRE11 activity not only reinstates gap repair in BRCA1-deficient cells, but also changes the pathway of gap repair in BRCA1-proficient cells. The second Aim will determine the mechanisms by which gaps are repaired in PARPi cells and test the innovative hypothesis that MRE11 processing is the key step that governs the choice between template switching and more error-prone translesion synthesis mechanisms of gap repair. Finally, we observed that, unlike the PARPi-sensitive BRCA1-deficient cells, ssDNA gaps are efficiently repaired when the same cells become PARPi-resistant. Thus, we will determine how MRE11 inhibition restores gap repair in BRCA1-deficient cells and whether targeting these repair pathways represents a novel strategy to overcome PARPi resistance in BRCA1-deficient cells. This knowledge is crucial for basic research to inform how nucleases control the balance between error-free and mutagenic mechanisms of ssDNA gap repair and how targeting these mechanisms modulates chemotherapy response in BRCA1-deficient tumors.
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