Date of Award
Summer 8-31-2025
Document Type
Campus Access Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Biology/Molecular, Cellular, and Organismal Biology
First Advisor
Shailja Pathania
Second Advisor
Changmeng Cai
Third Advisor
Alexey Veraksa, Andrew Grosovsky, Abby Green
Abstract
BRCA2 is a breast and ovarian cancer susceptibility gene that plays a major role in DNA damage repair in part by protecting the integrity of stalled replication forks. Mutations in BRCA2 are among the most common hereditary drivers of breast and ovarian cancer and significantly increases the lifetime cancer risk. Previous work from our group has shown that loss of BRCA2 impairs repair of stalled replication forks and leads to accumulation of single-stranded DNA (ssDNA) at stalled and collapsed replication forks. However, critical gaps remain in understanding how BRCA2 deficiency triggers fork collapse and how ssDNA accumulation in BRCA2-deficient cells leads to increased genomic instability remains unclear.
Using a novel uracil-in-DNA probe (U-DNA), we show that BRCA2 deficiency leads to an increased accumulation of uracil in ssDNA (U-ssDNA) at stalled forks. In keeping with high U-ssDNA accumulation, I observe an increase in abasic sites (AP-sites) upon replication stress in BRCA2-deficient cells. I identify APOBEC3 proteins (APOBEC3A and APOBEC3B) as key drivers of uracil accumulation in BRCA2-deficient cells through their cytosine deamination activity. Notably, replication stress induced by agents such as hydroxyurea or cisplatin, leads to upregulation of APOBEC3 proteins. Mechanistically, I show that ssDNA in BRCA2-deficient cells undergoing replication stress, serves as a trigger for the upregulation of A3A and A3B via the NF-kB pathway. Inhibition of the NF-kB signaling pathway using an inhibitor targeting both IKKα or IKKβ significantly downregulates A3A and A3B expression and decreases U-ssDNA levels. Interestingly, I find that unlike BRCA2-deficient cells, BRCA1-deficient cells do not accumulate AP-sites and do not upregulate A3A and/or A3B upon replication stress, highlighting the unique susceptibility of BRCA2-deficient cells to A3A/A3B upregulation.
I further demonstrate that sequential processing of AP sites in ssDNA by APE1 leads to stalled fork collapse, genomic instability and increased cell sensitivity to replication stress in BRCA2-deficient cells. Loss of A3A/3B or APE1 rescues genomic instability and cell viability in BRCA2-deficient cells undergoing replication stress. In keeping with this data, clinically, BRCA2-mutant ovarian tumors with low APE1 and/or high APOBEC and low APE1 levels correlate with worse prognosis. Overall, our findings establish upregulation of APOBEC3A/3B as a major driver of genomic instability in BRCA2 deficient tumors. Additionally, this work indicates that the levels of APOBEC3A/B and APE1 can influence BRCA2 mutant tumor evolution and response to therapy.
Recommended Citation
Situ, Kathy, "BRCA2 Deficiency and Replication Stress Drive APOBEC3-Mediated Genomic Instability" (2025). Graduate Doctoral Dissertations. 1107.
https://scholarworks.umb.edu/doctoral_dissertations/1107
Comments
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