DNA double-strand breaks are dangerous lesions that must be repaired for cells to survive. Recent studies have demonstrated that error-prone break repair processes such as alternative end joining operate when accurate repair pathways are compromised. These inaccurate processes lead to genomic deletions and chromosome translocations that are associated with genetic diseases and human cancers, especially lymphomas and leukemias. The goal of our research is to characterize molecular mechanisms of inaccurate double-strand break repair and to determine how their utilization may lead to disease. To accomplish this, we propose to elucidate how translesion DNA polymerases contribute to inaccurate repair in the model metazoan Drosophila melanogaster. Specifically, we will investigate a newly-discovered role for DNA polymerase theta in alternative end joining and test the hypothesis that polymerase theta uses coordinated helicase and polymerase activities to promote annealing at microhomologous sequences. Transgenic flies with domain-specific mutations in polymerase theta will be tested for their ability to support alternative end-joining repair. In addition, mutant and wild-type variants of the protein will be expressed in insect cells, purified, and tested in helicase and polymerase assays. In companion experiments, we will use in vivo reporter systems to define the roles of translesion polymerases eta, zeta, and Rev1 in homologous recombination repair of DNA double-strand breaks and gaps that result from I-SceI endonuclease expression and transposon excision. We will also compare the roles of replicative polymerases in break repair requiring various amounts of DNA synthesis and determine how their impairment affects the utilization of translesion polymerases in similar repair contexts. To gain insight into the ways in which local sequence context can affect alternative end joining, we will construct plasmids with I-SceI sites embedded in various DNA repeat contexts and assay repair in embryos expressing I- SceI. Finally, we will characterize a deletion-prone repair pathway that operates in the absence of polymerase theta-mediated alternative end joining and test the model that this backup pathway is a major cause of tumorigenesis in actively dividing tissues of adult flies. Together, these experiments will serve to define how translesion polymerases and associated proteins carry out inaccurate rejoining of broken DNA and will provide important insight into how error-prone double-strand break repair can cause genome instability frequently observed in cancer and related diseases.

Public Health Relevance

Cancer and other genetic diseases can be caused by changes that occur when both strands of a DNA double helix are broken and then repaired incorrectly. Surprisingly, certain processes that repair DNA breaks tend to be inaccurate. The goal of this proposal is to identify proteins involved in inaccurate repair and characterize how their action may lead to cellular changes that cause tumor formation.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Molecular Genetics B Study Section (MGB)
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Janes, Daniel E
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Tufts University
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United States
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Beagan, Kelly; Armstrong, Robin L; Witsell, Alice et al. (2017) Drosophila DNA polymerase theta utilizes both helicase-like and polymerase domains during microhomology-mediated end joining and interstrand crosslink repair. PLoS Genet 13:e1006813
Khodaverdian, Varandt Y; McVey, Mitch (2017) Rapid Detection of ?-H2Av Foci in Ex Vivo MMS-Treated Drosophila Imaginal Discs. Methods Mol Biol 1644:203-211
Alexander, Jessica L; Beagan, Kelly; Orr-Weaver, Terry L et al. (2016) Multiple mechanisms contribute to double-strand break repair at rereplication forks in Drosophila follicle cells. Proc Natl Acad Sci U S A 113:13809-13814
Beagan, Kelly; McVey, Mitch (2016) Linking DNA polymerase theta structure and function in health and disease. Cell Mol Life Sci 73:603-15
McVey, Mitch; Khodaverdian, Varandt Y; Meyer, Damon et al. (2016) Eukaryotic DNA Polymerases in Homologous Recombination. Annu Rev Genet 50:393-421
Rodgers, Kasey; McVey, Mitch (2016) Error-Prone Repair of DNA Double-Strand Breaks. J Cell Physiol 231:15-24
Kloosterman, Wigard P; Francioli, Laurent C; Hormozdiari, Fereydoun et al. (2015) Characteristics of de novo structural changes in the human genome. Genome Res 25:792-801
Bolterstein, Elyse; Rivero, Rachel; Marquez, Melissa et al. (2014) The Drosophila Werner exonuclease participates in an exonuclease-independent response to replication stress. Genetics 197:643-52
McVey, Mitch (2014) RPA puts the brakes on MMEJ. Nat Struct Mol Biol 21:348-9
Thomas, Adam M; Hui, Carrie; South, Adam et al. (2013) Common variants of Drosophila melanogaster Cyp6d2 cause camptothecin sensitivity and synergize with loss of Brca2. G3 (Bethesda) 3:91-9

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