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.
Identifer | oai:union.ndltd.org:ucf.edu/oai:stars.library.ucf.edu:honorstheses-1566 |
Date | 01 January 2019 |
Creators | Shah, Jil |
Publisher | STARS |
Source Sets | University of Central Florida |
Language | English |
Detected Language | English |
Type | text |
Format | application/pdf |
Source | Honors Undergraduate Theses |
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