The long-term objective of this proposal is to determine how the chromatin response to a mammalian DSB, mediated by the phosphorylation of histone H2AX, regulates DSB repair. We discovered a critical role for H2AX in controlling sister chromatid recombination (SCR), a homologous recombination (HR) process that operates during S and G2 phases of the cell cycle to repair replication-associated DNA damage in an error free manner. In recent work, we discovered that MDC1, an H2AX-binding adaptor protein, is the major mediator of H2AX-dependent HR/SCR. We discovered that the chromatin response to a mammalian DSB encodes additional DSB repair functions, regulating both single strand annealing (SSA) and non-homologous end joining (NHEJ). Our work revealed a previously unsuspected degree of specificity to chromatin-based DSB repair functions and established the existence of a """"""""histone code"""""""" for DSB repair. In this proposal, we will determine the biochemical basis of H2AX/MDC1-mediated HR/SCR, by analyzing a new MDC1-interacting protein complex involving TRIP12 and nucleophosmin (NPM1), which we have recently identified. We will also analyze other DSB repair functions encoded within the DSB chromatin response.
Our Specific Aims are: 1. Define DSB repair functions of TRIP12 and NPM1. We will use genetic, biochemical and cell biological techniques to accomplish this Aim. 2. Define roles of H2AX and associated proteins in SSA and in NHEJ. We have developed novel mammalian reporters for SSA and NHEJ that deliver rapid, specific and quantitative flow cytometric measurement of these DSB repair outcomes. We will use these reporters to determine the contribution of H2AX and its associated proteins to SSA and NHEJ. 3. Determine the mechanisms by which 53BP1 mediates NHEJ. We will use NHEJ reporters that we developed previously, as well as new reporters described herein, to determine how 53BP1 executes NHEJ. We will combine this with use of a novel multi-photon laser for DSB induction and real time imaging, as well as conventional DSB inducing agents in 53BP1-/- cells to study how 53BP1 regulates NHEJ. Success in the projects described in this proposal will: 1. Elucidate a chromatin-based pathway for regulating HR/SCR. 2. Identify TRIP12 and NPM1 as novel chromatin-associated components of mammalian HR. 3. Validate new, rapid and specific reporters of single strand annealing (SSA) and NHEJ. 4. Reveal how 53BP1 regulates NHEJ 5. Validate a novel multi-photon laser and real time imaging tool to study the kinetics of DSB repair.

Public Health Relevance

Histone H2AX, a component of the DNA-protein structure called chromatin, is important for preventing genomic instability and cancer in mammals. We discovered that H2AX is needed for the accurate repair of DNA breaks by a process called sister chromatid recombination. In this proposal, we will conduct experiments to find out how H2AX and its binding proteins in chromatin regulate sister chromatid recombination and other forms of DSB repair.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM073894-07
Application #
8499347
Study Section
Cancer Etiology Study Section (CE)
Program Officer
Janes, Daniel E
Project Start
2005-09-15
Project End
2015-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
7
Fiscal Year
2013
Total Cost
$315,885
Indirect Cost
$123,346
Name
Beth Israel Deaconess Medical Center
Department
Type
DUNS #
071723621
City
Boston
State
MA
Country
United States
Zip Code
02215
Willis, Nicholas A; Rass, Emilie; Scully, Ralph (2015) Deciphering the Code of the Cancer Genome: Mechanisms of Chromosome Rearrangement. Trends Cancer 1:217-230
Elia, Andrew E H; Wang, David C; Willis, Nicholas A et al. (2015) RFWD3-Dependent Ubiquitination of RPA Regulates Repair at Stalled Replication Forks. Mol Cell 60:280-93
Liu, Pengda; Gan, Wenjian; Guo, Chunguang et al. (2015) Akt-mediated phosphorylation of XLF impairs non-homologous end-joining DNA repair. Mol Cell 57:648-61
Liu, Xue-Song; Chandramouly, Gurushankar; Rass, Emilie et al. (2015) LRF maintains genome integrity by regulating the non-homologous end joining pathway of DNA repair. Nat Commun 6:8325
Beck, Carole; Boehler, Christian; Guirouilh Barbat, Josée et al. (2014) PARP3 affects the relative contribution of homologous recombination and nonhomologous end-joining pathways. Nucleic Acids Res 42:5616-32
Willis, Nicholas A; Chandramouly, Gurushankar; Huang, Bin et al. (2014) BRCA1 controls homologous recombination at Tus/Ter-stalled mammalian replication forks. Nature 510:556-9
Hu, Yiduo; Petit, Sarah A; Ficarro, Scott B et al. (2014) PARP1-driven poly-ADP-ribosylation regulates BRCA1 function in homologous recombination-mediated DNA repair. Cancer Discov 4:1430-47
Liu, Yan; Marks, Kevin; Cowley, Glenn S et al. (2013) Metabolic and functional genomic studies identify deoxythymidylate kinase as a target in LKB1-mutant lung cancer. Cancer Discov 3:870-9
Somyajit, Kumar; Basavaraju, Shivakumar; Scully, Ralph et al. (2013) ATM- and ATR-mediated phosphorylation of XRCC3 regulates DNA double-strand break-induced checkpoint activation and repair. Mol Cell Biol 33:1830-44
Chandramouly, Gurushankar; Kwok, Amy; Huang, Bin et al. (2013) BRCA1 and CtIP suppress long-tract gene conversion between sister chromatids. Nat Commun 4:2404

Showing the most recent 10 out of 31 publications