When cells undergo mitosis, they must replicate all six billion base pairs of DNA within the nucleus. With the sheer volume of information, it is impossible to replicate with 100% accuracy each time. Homologous recombination (HR) is one of many mechanisms the body has developed to correct and repair replication errors to DNA. HR is specific to double-stranded breaks to DNA, and it requires a sister chromatid to preserve the genetic code. BRCA1 and Rbbp8 interact to form a complex that is heavily involved in this process. Although there is a strong consensus about the involvement of these proteins in a cell cycle-dependent manner, there are discrepancies in the current literature regarding when homologous recombination repair occurs. The goal of this thesis is to elucidatethe true activity of BRCA1-Rbbp8 complex in the HR process. First, a meta-analysis was performed to review current research to understand the various experimental protocols that led to the conflicting conclusions about cell-cycle activity of HR. Then, we measured the mRNA levels of BRCA1 and Rbbp8 during different phases of the cell cycle.In order to accomplish this, the cell cycle of the L cells was synchronized using thymidine and RO-3306 for the S and G2phases, respectively. The RNA was collected and converted to cDNA via reverse transcriptase. Next, RT-qPCR was performed to measure the expression of BRCA1 and Rbbp8. The levels of Rbbp8 are not significantly different between these phases, though there was a clear downward trend worth noting. Furthermore, a statistically significant increase in BRCA1 as the cells moved from S phase to G2phase was observed. When normalized tothe housekeeping gene, GAPDH, the levels of mRNA for BRCA1 during the S phase were significant lower than the control group. These results suggest that HR occurs during both S and G2, but BRCA1 and Rbbp8 interact only during G2.
Bachelor of Science (B.S.)
College of Medicine
Burnett School of Biomedical Sciences
Shah, Jil, "Investigation of the Cell-Cycle Dependent Activity of the BRCA1-Rbbp8 Complex for Homologous Recombination" (2019). Honors Undergraduate Theses. 527.