Mutations in the gene encoding the ATM serine-threonine kinase cause Ataxia Telangiectasia (A-T), a disease marked by lymphopenia and an increased incidence of lymphoid tumors with translocations involving antigen receptor loci, suggesting that ATM functions during V(D)J recombination. ATM activates cell cycle checkpoints in response to DNA double strand breaks (DSBs). However, the lymphoid phenotypes of A-T are not recapitulated in mice with isolated deficiencies in checkpoint pathways. We have demonstrated that ATM functions to repair DSBs generated during V(D)J recombination, and to suppress the aberrant resolution of these DSBs as chromosomal translocations. The combined defect in checkpoint pathways and DSB repair explains some of the lymphoid phenotypes of A-T. Given all of the defects in V(D)J recombination observed in ATM-deficient lymphocytes, we have proposed that ATM functions, in part, to maintain the stability of DSB complexes after RAG-mediated DNA cleavage, which is a hypothesis that will be directly tested in Specific Aim One. Although ATM could function directly in the repair of RAG-mediated DSBs, we expect that ATM will likely phosphorylate proteins that perform this function. In this regard we show that the MRN complex (Mre11, Rad50 and Nbs1), 53BP1 and H2AX, which are all targets of ATM, function in the response to RAG-mediated DSBs. How these proteins function in the ATM-dependent pathway of RAG-DSB repair will be elucidated in Specific Aim Two. In addition, we will consider the possibility that the RAG proteins may have ATM-dependent functions in the joining step of the V(D)J recombination reaction. Importantly, we believe that these different proteins will function in an integrated manner in the ATM-dependent RAG-DSB repair pathway. Finally, we will investigate the mechanisms by which DNA DSBs generated during V(D)J recombination in ATM-deficient cells become aberrantly resolved as chromosomal translocations (Specific Aim 3). The completion of these aims will provide important new information about: 1) how RAG-mediated DSBs are repaired;2) how ATM functions in DSB repair and in maintaining genomic stability;and 3) the mechanisms that promote the formation of chromosomal translocations.

Public Health Relevance

The ataxia telangiectasia mutated (ATM) protein is a critical initiator of DNA damage responses. We have shown that ATM is also involved in the repair of RAG-mediated DSBs generated during lymphocyte antigen receptor gene assembly. Here we propose to identify the proteins in the ATM-dependent pathway of RAG- DSB repair and determine how they function. Furthermore, we will elucidate the mechanisms that lead to the frequent aberrant resolution of DNA breaks as chromosomal translocations in ATM-deficient cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI074953-04
Application #
8271430
Study Section
Cellular and Molecular Immunology - A Study Section (CMIA)
Program Officer
Nasseri, M Faraz
Project Start
2008-12-02
Project End
2013-11-30
Budget Start
2011-12-01
Budget End
2012-11-30
Support Year
4
Fiscal Year
2012
Total Cost
$372,438
Indirect Cost
$127,413
Name
Washington University
Department
Pathology
Type
Schools of Medicine
DUNS #
068552207
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
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Dorsett, Yair; Zhou, Yanjiao; Tubbs, Anthony T et al. (2014) HCoDES reveals chromosomal DNA end structures with single-nucleotide resolution. Mol Cell 56:808-18
Makharashvili, Nodar; Tubbs, Anthony T; Yang, Soo-Hyun et al. (2014) Catalytic and noncatalytic roles of the CtIP endonuclease in double-strand break end resection. Mol Cell 54:1022-33
Lee, Baeck-Seung; Gapud, Eric J; Zhang, Shichuan et al. (2013) Functional intersection of ATM and DNA-dependent protein kinase catalytic subunit in coding end joining during V(D)J recombination. Mol Cell Biol 33:3568-79
Um, Jee-Hyun; Brown, Alexandra L; Singh, Samarendra K et al. (2013) Metabolic sensor AMPK directly phosphorylates RAG1 protein and regulates V(D)J recombination. Proc Natl Acad Sci U S A 110:9873-8
Helmink, Beth A; Sleckman, Barry P (2012) The response to and repair of RAG-mediated DNA double-strand breaks. Annu Rev Immunol 30:175-202
Bednarski, Jeffrey J; Nickless, Andrew; Bhattacharya, Deepta et al. (2012) RAG-induced DNA double-strand breaks signal through Pim2 to promote pre-B cell survival and limit proliferation. J Exp Med 209:11-7
Helmink, Beth A; Tubbs, Anthony T; Dorsett, Yair et al. (2011) H2AX prevents CtIP-mediated DNA end resection and aberrant repair in G1-phase lymphocytes. Nature 469:245-9
Gapud, Eric J; Dorsett, Yair; Yin, Bu et al. (2011) Ataxia telangiectasia mutated (Atm) and DNA-PKcs kinases have overlapping activities during chromosomal signal joint formation. Proc Natl Acad Sci U S A 108:2022-7
Gapud, Eric J; Lee, Baeck-Seung; Mahowald, Grace K et al. (2011) Repair of chromosomal RAG-mediated DNA breaks by mutant RAG proteins lacking phosphatidylinositol 3-like kinase consensus phosphorylation sites. J Immunol 187:1826-34

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