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.

Public Health Relevance

This proposal aims to understand the molecular mechanisms and regulation of DNA recombination, an essential DNA repair pathway preventing genome instability and cancer. Both the mechanism and proteins mediating DNA repair processes are conserved in evolution, so our proposed research in the model organism 2. SPECIFIC AIMS Homologous recombination (HR) is a conserved process that repairs spontaneous and induced DNA doublestrand breaks (DSBs) and gaps. Defects in HR lead to genome destabilization and disease, including cancer. While HR is essential for DSB repair and usually restores the original sequence at the site of the break, its unrestrained usage could also lead to genome rearrangements. Our long-term goal is to understand the genetic requirements, molecular mechanisms, and regulation of distinct HR pathways. A key reaction of HR is DNA synthesis that copies homologous sequences from a donor template. In our continuation project, we will focus on the mechanism and control of repair DNA synthesis during HR events. The repair DNA synthesis will be examined within the context of (1) the repair of two-ended DSBs by gene conversion (GC), where both 3'ends prime DNA synthesis and thus only short leading strands are synthesized, and (2) break induced replication (BIR), in which a single DSB end invades a donor template, followed by extensive leading and lagging DNA synthesis. Both pathways play distinct yet critical roles in cells. We postulate that DNA synthesis is tightly regulated in cells to accomplish timely repair. Our specific aims are to: 1. Identify and characterize enzymes that promote DNA synthesis during recombination. A fundamental gap in understanding HR is the step of repair-specific DNA synthesis. It remains unclear which polymerases are involved and whether there are any helicases functioning in DNA synthesis. Using chromatin immunoprecipitation (ChIP) we found that three polymerases Pol?, Pol? and Pol? are recruited to DSBs during gene conversion. We will define the unique and redundant roles of these polymerases and how the choice of polymerase is regulated. We present evidences that Pif1 is the first repair-specific helicase functioning in DNA synthesis in eukaryotes. Pif1 acts together with Pol? in repair DNA synthesis with significant impacts on error rate. Pif1 is very important for BIR and crossover pathway. Purified Pif1 protein greatly stimulates DNA synthesis by Pol? in a reconstituted HR reaction mediated by the Rad51 recombinase. Using a combination of genetic, molecular and biochemical approaches we aim to understand how Pif1 stimulates DNA synthesis at the molecular level, to identify its interactors and its role in mutagenesis, and to understand the consequences of Pif1 deletion on DSB repair. Besides Pif1, the Mcm2-7 replicative helicase, and almost all replication proteins were proposed to be involved in BIR. The experiments presented herein will illuminate whether and why two helicases are needed, will distinguish whether they work sequentially or cooperatively during BIR, and how the replicative helicase Mcm2-7, which is subjected to tight cell cycle control, is activated at DSBs in G2/M cells to promote BIR. Results of this aim will (1) reveal novel roles of several proteins including the first non- replicative DNA helicase in eukaryotes that functions in repair DNA synthesis, (2) provide a comprehensive model of DNA synthesis during DSB repair, (3) furnish a new model of DNA mutagenesis during DSB repair. 2. Understand the mechanism of BIR. Using an isotope density transfer assay we will establish whether DNA synthesis in BIR is semi-conservative as in normal replication or conservative. Our preliminary data suggest that Pif1 helicase stimulates DNA bubble migration suggesting a conservative mode of DNA synthesis. We will test the role of Pif1, Mcm2-7, GINS and resolvases that could cleave the branched DNA structures formed after initial strand invasion in determining DNA synthesis mode in BIR. Results of this aim will provide physical demonstration of the BIR mechanism in vivo, will distinguish existing BIR models. Given the outstanding conservation of the proteins investigated, our work is relevant to delineating the mechanisms of HR-mediated DNA in human cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM080600-06A1
Application #
8437897
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
2013-06-01
Budget End
2014-04-30
Support Year
6
Fiscal Year
2013
Total Cost
$295,513
Indirect Cost
$106,687
Name
Baylor College of Medicine
Department
Genetics
Type
Schools of Medicine
DUNS #
051113330
City
Houston
State
TX
Country
United States
Zip Code
77030
Wilson, Marenda A; Kwon, YoungHo; Xu, Yuanyuan et al. (2013) Pif1 helicase and Polýý promote recombination-coupled DNA synthesis via bubble migration. Nature 502:393-6
Malkova, Anna; Ira, Grzegorz (2013) Break-induced replication: functions and molecular mechanism. Curr Opin Genet Dev 23:271-9
Saini, Natalie; Ramakrishnan, Sreejith; Elango, Rajula et al. (2013) Migrating bubble during break-induced replication drives conservative DNA synthesis. Nature 502:389-92
Peng, Guang; Dai, Hui; Zhang, Wei et al. (2012) Human nuclease/helicase DNA2 alleviates replication stress by promoting DNA end resection. Cancer Res 72:2802-13
Oum, Ji-Hyun; Seong, Changhyun; Kwon, Youngho et al. (2011) RSC facilitates Rad59-dependent homologous recombination between sister chromatids by promoting cohesin loading at DNA double-strand breaks. Mol Cell Biol 31:3924-37
Chen, Xuefeng; Niu, Hengyao; Chung, Woo-Hyun et al. (2011) Cell cycle regulation of DNA double-strand break end resection by Cdk1-dependent Dna2 phosphorylation. Nat Struct Mol Biol 18:1015-9
Shim, Eun Yong; Chung, Woo-Hyun; Nicolette, Matthew L et al. (2010) Saccharomyces cerevisiae Mre11/Rad50/Xrs2 and Ku proteins regulate association of Exo1 and Dna2 with DNA breaks. EMBO J 29:3370-80
Chung, Woo-Hyun; Zhu, Zhu; Papusha, Alma et al. (2010) Defective resection at DNA double-strand breaks leads to de novo telomere formation and enhances gene targeting. PLoS Genet 6:e1000948
Hastings, P J; Ira, Grzegorz; Lupski, James R (2009) A microhomology-mediated break-induced replication model for the origin of human copy number variation. PLoS Genet 5:e1000327
Hastings, P J; Lupski, James R; Rosenberg, Susan M et al. (2009) Mechanisms of change in gene copy number. Nat Rev Genet 10:551-64

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