Errors made during V(D)J recombination, the process that assembles antigen receptor genes, can lead to chromosomal translocations and the development of human malignancies, particulariy childhood leukemias. One important cause of such translocations is the improper targeting of the RAG1 and RAG2 proteins, which constitute the central components of the V(D)J recombination machinery. In the first phase of V(D)J recombination, DNA substrate recognition and cleavage take place in highly organized nucleoprotein complexes whose integrity and specificity are determined by f^GI and RAG2, probably in conjunction with the DNA bending protein HMGB1. Little is known about the structure of these complexes, the conformational changes that occur during DNA binding, or the regulation of complex formation in vivo. Using novel biochemical, biophysical, and in vivo methodologies developed for the study of the RAG proteins during the previous funding period, three major aims will be pursued: 1) To understand the structure and dynamics of RAG-DNA complexes and determine how structural changes relate to RAG function;work under this aim will result in refined three-dimensional models of the DNA in fRAG-DNA complexes as well as the characterization of the pathway leading to complex formation. 2) To identify the role of HMGB1 in DNA binding and cleavage by RAG;work under this aim will take advantage of fluorescently-labeled HMGB1 and will reveal the stoichiometry, positioning, and function of HMGB1 in RAG-DNA complexes, as well as its contribution to the targe DNA bends identified in the prior funding period. 3) To determine the patterns of RAG1 and RAG2 binding throughout the genome of mouse and human developing lymphocytes;work under this aim will lead to an understanding of the rules that govern RAG recruitment to """"""""recombination centers"""""""" in antigen receptor loci and to the thousands of sites they occupy elsewhere in the genome. Structural information from aims 1 and 2 will be connected to function through the use of altered DNA substrates and mutant RAG and HI ^GB1 proteins, and will be used to formulate models of how DNA recognition occurs within the genome. These models will be tested through in vivo binding experiments in developing lymphocytes and in cell line models. These studies should provide insights into how the RAG proteins and DNA communicate with one another at the molecular level and how this interaction leads both to carefully orchestrated antigen receptor gene assembly and to mis-recognition errors that underlie genome instability.

Public Health Relevance

; These experiments study V(D)J recombination, an essential process that occasionally makes mistakes and generates aberrations in human chromosomes during the development of white blood cells known as lymphocytes. These aberrations contribute to the development of certain blood cancers and it is hoped that this work will provide insights into the causes of these mistakes and methods by which they could be prevented.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37AI032524-23
Application #
8660578
Study Section
Special Emphasis Panel (NSS)
Program Officer
Nasseri, M Faraz
Project Start
1992-04-01
Project End
2017-05-31
Budget Start
2014-06-01
Budget End
2015-05-31
Support Year
23
Fiscal Year
2014
Total Cost
$374,625
Indirect Cost
$149,625
Name
Yale University
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
Fisher, Megan R; Rivera-Reyes, Adrian; Bloch, Noah B et al. (2017) Immature Lymphocytes Inhibit Rag1 and Rag2 Transcription and V(D)J Recombination in Response to DNA Double-Strand Breaks. J Immunol 198:2943-2956
Carmona, Lina Marcela; Schatz, David G (2017) New insights into the evolutionary origins of the recombination-activating gene proteins and V(D)J recombination. FEBS J 284:1590-1605
Huang, Shengfeng; Tao, Xin; Yuan, Shaochun et al. (2016) Discovery of an Active RAG Transposon Illuminates the Origins of V(D)J Recombination. Cell 166:102-14
Maman, Yaakov; Teng, Grace; Seth, Rashu et al. (2016) RAG1 targeting in the genome is dominated by chromatin interactions mediated by the non-core regions of RAG1 and RAG2. Nucleic Acids Res 44:9624-9637
Carmona, Lina Marcela; Fugmann, Sebastian D; Schatz, David G (2016) Collaboration of RAG2 with RAG1-like proteins during the evolution of V(D)J recombination. Genes Dev 30:909-17
Brauer, Patrick M; Pessach, Itai M; Clarke, Erik et al. (2016) Modeling altered T-cell development with induced pluripotent stem cells from patients with RAG1-dependent immune deficiencies. Blood 128:783-93
Shetty, Keerthi; Schatz, David G (2015) Recruitment of RAG1 and RAG2 to Chromatinized DNA during V(D)J Recombination. Mol Cell Biol 35:3701-13
Lovely, Geoffrey A; Brewster, Robert C; Schatz, David G et al. (2015) Single-molecule analysis of RAG-mediated V(D)J DNA cleavage. Proc Natl Acad Sci U S A 112:E1715-23
Hu, Jiazhi; Zhang, Yu; Zhao, Lijuan et al. (2015) Chromosomal Loop Domains Direct the Recombination of Antigen Receptor Genes. Cell 163:947-59
Mandal, Malay; Hamel, Keith M; Maienschein-Cline, Mark et al. (2015) Histone reader BRWD1 targets and restricts recombination to the Igk locus. Nat Immunol 16:1094-103

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