DNA recombination is an essential process that repairs DNA double-strand breaks (DSBs) and gaps that occur spontaneously, or are induced by chemicals or irradiation. In human, defects in recombination result in immune deficiencies, infertility, neurodegenerative disorders, developmental abnormalities, aging and cancer. DSBs as the most cytotoxic lesions are a fundamental component of the most prevalent cancer treatments, radiotherapy and radiomimetic chemotherapy. Therefore defining the genetic requirements and mechanisms of recombination pathways is of critical importance. A fundamental reaction during repair of broken chromosomes by recombination is DNA synthesis that copies homologous sequences from a template DNA molecule. The goal of this project is to understand the mechanisms and regulation of DNA synthesis during recombination, which remains very poorly understood. Two assays will be utilized to examine repair DNA synthesis that reflect two major recombination pathways with distinct DNA synthesis features. Both pathways play different yet important roles in cells. One is the simple repair of two-ended DSBs by gene conversion where both 3'ends prime DNA synthesis and thus only short leading strands are synthesized. The second assay employs break induced replication (BIR), in which a single DSB end invades a template, followed by extensive leading- and lagging- strand DNA synthesis. BIR is thought to be a mechanism of HR-dependent telomere maintenance in the absence of telomerase found in 10- 15% of all cancers.
The specific aims are: (1) To understand the unique and redundant functions of multiple DNA polymerases recruited to DSBs and determine which DNA helicases and other enzymes specifically promote DNA synthesis during homologous recombination. The major focus will be on studying Pif1, the DNA helicase that we propose to be the first eukaryotic nonreplicative helicase that stimulates DNA synthesis during recombination. (2) To understand the mechanism of Break Induced Replication. Using isotope density transfer we will establish the mode of DNA synthesis in BIR and determine the role of DNA helicases and structure specific nucleases in BIR. Together we will provide a comprehensive view of DNA synthesis during homologous recombination.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM080600-07
Application #
8667465
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Janes, Daniel E
Project Start
2007-05-01
Project End
2017-04-30
Budget Start
2014-05-01
Budget End
2015-04-30
Support Year
7
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Baylor College of Medicine
Department
Genetics
Type
Schools of Medicine
DUNS #
City
Houston
State
TX
Country
United States
Zip Code
77030
Lopez, Christopher R; Singh, Shivani; Hambarde, Shashank et al. (2017) Yeast Sub1 and human PC4 are G-quadruplex binding proteins that suppress genome instability at co-transcriptionally formed G4 DNA. Nucleic Acids Res 45:5850-5862
Miller, Adam S; Daley, James M; Pham, Nhung Tuyet et al. (2017) A novel role of the Dna2 translocase function in DNA break resection. Genes Dev 31:503-510
Elango, Rajula; Sheng, Ziwei; Jackson, Jessica et al. (2017) Break-induced replication promotes formation of lethal joint molecules dissolved by Srs2. Nat Commun 8:1790
Kumar, S; Peng, X; Daley, J et al. (2017) Inhibition of DNA2 nuclease as a therapeutic strategy targeting replication stress in cancer cells. Oncogenesis 6:e319
Lema├žon, Delphine; Jackson, Jessica; Quinet, Annabel et al. (2017) MRE11 and EXO1 nucleases degrade reversed forks and elicit MUS81-dependent fork rescue in BRCA2-deficient cells. Nat Commun 8:860
Chen, Xuefeng; Niu, Hengyao; Yu, Yang et al. (2016) Enrichment of Cdk1-cyclins at DNA double-strand breaks stimulates Fun30 phosphorylation and DNA end resection. Nucleic Acids Res 44:2742-53
Carvalho, Claudia M B; Pfundt, Rolph; King, Daniel A et al. (2015) Absence of heterozygosity due to template switching during replicative rearrangements. Am J Hum Genet 96:555-64
Xue, Xiaoyu; Choi, Koyi; Bonner, Jaclyn N et al. (2015) Selective modulation of the functions of a conserved DNA motor by a histone fold complex. Genes Dev 29:1000-5
Mayle, Ryan; Campbell, Ian M; Beck, Christine R et al. (2015) DNA REPAIR. Mus81 and converging forks limit the mutagenicity of replication fork breakage. Science 349:742-7
Sakofsky, Cynthia J; Ayyar, Sandeep; Deem, Angela K et al. (2015) Translesion Polymerases Drive Microhomology-Mediated Break-Induced Replication Leading to Complex Chromosomal Rearrangements. Mol Cell 60:860-72

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