Duke University Durham, North Carolina, United States
Abstract: BRCA1 and BRCA2 are tumor-suppressor genes that play critical roles in DNA homologous recombination and repair. Deficiency in BRCA1 and BRCA2, caused by mutations in these genes, are frequently associated with breast, ovarian, and other cancers.
While homozygous BRCA1/2 mutations are known to promote genomic instability and tumorigenesis by compromising the HR pathway, heterozygous mutations do not impair HR, and their contribution to cancer development remains unclear. Since BRCA1 and BRCA2 proteins are involved in DNA replication, we hypothesize that heterozygous mutations in their genes may increase replication stress. Using the DNA fiber assay, we examined replication dynamics in cells expressing either wild-type BRCA1/2 or heterozygous mutations in one of these genes. We found that the heterozygous mutant strains produced shorter DNA fibers, indicating that the mutations impaired DNA replication. Moreover, treatment with nuclease S1 during the assay further decreased fiber lengths in mutant cells, suggesting an accumulation of single-stranded DNA (ssDNA) gaps. Together, our study shows that heterozygous BRCA1/2 mutations indeed increase replication stress, providing new insights into their molecular function in DNA replication and suggesting a potential mechanism by which they promote cancer development.
In addition to studying the BRCA1/2 heterozygous mutations, I plan to use a stem cell model to identify genetic interactors of BRCA2 to better understand its function. BRCA2 deletion in embryonic stem cells (ESCs) causes lethality due to impaired DNA homologous recombination. It was recently reported that the deletion of another gene, PTIP, rescues this lethality by protecting BRCA2-deficient cells from DNA damage. Based on these findings, I propose to carry out a CRISPR knockout-based genetic suppressor screen in BRCA2 conditional deletion ESCs in order to find additional factors that are involved in BRCA2-mediated DNA damage repair pathways.
Together, we hope our research will help to better understand the underlying mechanisms behind BRCA1/2 mutation-associated cancers and identify potential future therapeutic targets.
Funding Source: This research project is supported by a grant from the National Cancer Institute to Dr. Lee Zou (CA263934) at Duke University
