Abstract

Single-stranded (ss) DNA gaps are potentially genome-destabilizing structures that require prompt repair (or filling) to prevent DNA breakage and genome instability. We previously showed that gap repair is defective in cancer cells with deficiencies in the breast cancer susceptibility gene BRCA1. These findings explain why ssDNA gaps accumulate in BRCA1-deficient cells in response to therapies that target DNA, such as platinum-based compounds, or therapies that inhibit specific repair pathways, such as PARP inhibitors (PARPi). We also discovered 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 metabolism. While processing of DNA double-stranded breaks has been widely studied, the corresponding molecular details are missing for ssDNA gaps. This thesis begins by first elucidating the molecular mechanism that regulates ssDNA gap processing in BRCA1-proficient and -deficient cells by combining electron microscopy and single-molecule DNA fiber approaches with in vitro biochemical assays. We discovered that MRE11 resects the 3’ end of the ssDNA gaps, whereas the long-range resection nucleases EXO1 and DNA2 process the 5’ end. We also discovered that DNA2 works in concert with either BLM or WRN and that both the EXO1 and the DNA2/BLM or DNA2/WRN resection pathways are utilized to resect the 5’ end of the gap. Importantly, we also found that this nuclease resection pathway is deregulated in BRCA1-deficient cells, leading to larger ssDNA gaps compared to BRCA1-proficient cells. These larger ssDNA gaps cannot be efficiently repaired by the canonical gap repair pathways and are converted into double-stranded breaks, leading to genomic instability. Our studies provide the first complete model for ssDNA gap resection following treatment with PARPi or DNA-damaging agents. This is critical to explain why ssDNA gaps cannot be repaired in BRCA1-deficient cells and to better understand the molecular mechanisms that modulate PARPi response in BRCA1-deficient tumors. Lastly, we investigated whether factors involved the ssDNA gap resection pathway are mutated in BRCA1-deficient cancers. In collaboration with Dr. Cynthia Ma, we combed through approximately 50 BRCA-mutated patient’s data who were treated with PARPi, providing the framework for future analysis.

Committee Chair

Alessandro Vindigni

Committee Members

Jieya Shao; Peter Burgers; Cynthia Ma; Zhongshen You

Degree

Doctor of Philosophy (PhD)

Author's Department

Biology & Biomedical Sciences (Cancer Biology)

Author's School

Graduate School of Arts and Sciences

Document Type

Dissertation

Date of Award

8-18-2025

Language

English (en)

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