The long-term objective of this research is to understand the factors and pathways that influence the extent of genetic loss during homologous repair of chromosomal breaks in mammalian cells. For this, we are studying the repair of a break generated by the rare cutting endonuclease, l-Scel. We have recently shown that deficiencies in a number of genes result in relatively more genetic loss during homologous repair of such breaks. We hypothesize that such effects on genetic loss could be due to the disruption in the control of discrete steps of homologous repair. One such step could be 5' to 3' DNA end resection, both since single stranded tails generated by this resection have the potential to promote more mutagenic repair pathways, and since single stranded tails appear to be important for DNA damage-induced cell cycle checkpoints. Replication during gene conversion is another important mechanistic step that could influence the extent of genetic loss during repair by determining the amount of genetic information transferred during gene conversion. We propose to test the hypothesis that the mechanistic control of the 5' to 3' resection and/or replication steps of homologous repair are critical for limiting genetic loss during chromosomal break repair.
The specific aims are: 1. To test the hypothesis that limiting 5' to 3' resection and/or replication is important to suppress genetic loss during gene conversion. For this, we will develop and analyze a series of recombination reporters, which differ in the degree of resection versus replication required for the repair event. 2. To test the hypothesis that individual genetic factors may influence genetic loss by affecting the control of resection and/or replication. For this, we propose to analyze the reporters described in Aim 1 in cells deficient for RAD51, BRCA1, and BRCA2. 3. To physically determine the frequency and extent of 5' to 3' resection of a chromosomal break in a variety of genetic contexts in mammalian cells. These physical experiments are fundamental to an understanding of how the control of resection may influence genetic loss. Relevance to public health: Our objective is to understand how damaged DNA is repaired, since failures in this process results in loss of genetic information. This objective is important for understanding the process of genetic loss 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. ? ? ?

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Molecular Genetics C Study Section (MGC)
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Pelroy, Richard
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City of Hope/Beckman Research Institute
United States
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Gu, Long; Lingeman, Robert; Yakushijin, Fumiko et al. (2018) The Anticancer Activity of a First-in-class Small-molecule Targeting PCNA. Clin Cancer Res 24:6053-6065
Mendez-Dorantes, Carlos; Bhargava, Ragini; Stark, Jeremy M (2018) Repeat-mediated deletions can be induced by a chromosomal break far from a repeat, but multiple pathways suppress such rearrangements. Genes Dev 32:524-536
Bhargava, Ragini; Sandhu, Manbir; Muk, Sanychen et al. (2018) C-NHEJ without indels is robust and requires synergistic function of distinct XLF domains. Nat Commun 9:2484
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Onyango, David O; Lee, Gabriella; Stark, Jeremy M (2017) PRPF8 is important for BRCA1-mediated homologous recombination. Oncotarget 8:93319-93337
Onyango, David O; Howard, Sean M; Neherin, Kashfia et al. (2016) Tetratricopeptide repeat factor XAB2 mediates the end resection step of homologous recombination. Nucleic Acids Res 44:5702-16
Morales, Maria E; Derbes, Rebecca S; Ade, Catherine M et al. (2016) Heavy Metal Exposure Influences Double Strand Break DNA Repair Outcomes. PLoS One 11:e0151367
Bhargava, Ragini; Onyango, David O; Stark, Jeremy M (2016) Regulation of Single-Strand Annealing and its Role in Genome Maintenance. Trends Genet 32:566-575
Skrdlant, Lindsey; Stark, Jeremy M; Lin, Ren-Jang (2016) Myelodysplasia-associated mutations in serine/arginine-rich splicing factor SRSF2 lead to alternative splicing of CDC25C. BMC Mol Biol 17:18
Kuo, Ching-Ying; Li, Xu; Stark, Jeremy M et al. (2016) RNF4 regulates DNA double-strand break repair in a cell cycle-dependent manner. Cell Cycle 15:787-98

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