Jeremy Stark, Ph.D. Research
Mechanistic Steps of Homologous DNA Repair in Mammalian Cells
While high-fidelity repair of DNA damage is essential for genome maintenance, individual DNA repair pathways can be prone to cause genetic loss. The long-term objective of our lab’s research is to understand the factors and pathways that influence the extent of genetic loss during repair of chromosomal breaks in mammalian cells. This objective is important for an understanding of the process of mutagenesis during cancer development, as well as for a mechanistic characterization of potential targets of drugs that could increase the efficacy of cancer treatments that utilize DNA damaging agents.
As one approach to this objective, we are studying the mechanism of homologous repair of a break generated by the rare cutting endonuclease, I-SceI. Specifically, we generate a mammalian cell line that contains an I-SceI site integrated into the chromosome in the context of a reporter gene, express I-SceI in the cells, and allow the cells to repair the break.  We then monitor the different products of such repair, which are associated with varying degrees of genetic loss. Furthermore, we are testing how disruption of individual genetic factors influences the efficiency of individual repair products.
For example, we found that disruption of RAD51 function by expression of a dominant-negative peptide, RAD51-K133R, resulted in a decrease in the efficiency of a precise form of repair, gene conversion, and an increase in two mutagenic forms of repair, single-strand annealing and extensive gene conversion. In addition, we found that the breast cancer susceptibility factors, BRCA1 and BRCA2, differentially affected these pathways. Disruption of BRCA2 caused a similar result as disruption of RAD51, in that the efficiency of gene conversion was decreased and single-strand annealing was increased. In contrast, disruption of BRCA1 caused a decrease in the efficiency of both gene conversion and single-strand annealing.
Recently, we have been analyzing a pathway of repair that results in deletion mutations, often with evidence of short homologous sequences at the repair junctions (alt-NHEJ). Such repair events mimic a variety of oncogenic genome rearrangements. We have begun to study the genetic factors that affect these events, and have found that KU/CtIP appear to affect the initial stages of repair of both alt-NHEJ and recombination. However, these pathways appear to diverge at later steps, as relates to the role of the repair factors RAD52, ERCC1, and RAD51. We are working to further characterize the mechanisms of these factors and pathways of chromosome break repair, in order to improve our understanding of the process of genetic loss during cancer development and treatment.