V(D)J recombination assembles the variable portions of immunoglobulin and T cell receptor genes and is essential for lymphocyte development. Errors in the recombination process can lead to chromosomal translocations and the development of human malignancies, particularly childhood leukemias. The causes of such translocations are thought to be improper targeting of the recombination machinery and the premature release of broken chromosomal ends before they have been properly rejoined. The proteins encoded by the recombination activating genes, RAG1 and RAG2, play central roles in targeting and DNA end rejoining during V(D)J recombination and are therefore important in the generation of translocations. In the first phase of V(D)J recombination, DNA substrate recognition and the production of double strand breaks take place in highly organized nucleoprotein complexes whose integrity and specificity are in large part determined by RAG1 and RAG2, probably in conjunction with the DNA bending protein HMGB1. Little is known about the structure of these complexes or the conformational changes that occur during DNA binding and cleavage. The hypothesis underlying this proposal is that the sequential, properly regulated formation of these complexes requires an orchestrated series of structural changes in both the substrate DNA and the RAG/HMGB1 proteins. We have demonstrated that RAG1 undergoes a conformational change when it binds DNA and have used fluorescence resonance energy transfer (FRET) assays to characterize DNA organization and bending in single substrate complexes as well as in higher order synaptic complexes. We propose to use a combination of biochemical and fluorescence methods to elucidate protein and DNA structural changes that occur during assembly of RAG protein-DNA complexes and to construct initial three- dimensional models of the DNA in these complexes. The structural information will be connected to function (catalysis of DNA cleavage) through the use of altered DNA substrates, mutated RAG proteins, and fluorescently labeled HMGB1. We are particularly interested in understanding the structural underpinnings of the requirement for asymmetric DNA substrates and the molecular rules governing recently discovered restrictions on the rearrangements of endogenous gene segments. We anticipate that these studies will provide insights into how the DNA and proteins communicate with and influence one another to create a complex within which properly coordinated cleavage events can occur. 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.

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 #
5R37AI032524-20
Application #
8082640
Study Section
Special Emphasis Panel (ZRG1-IMM-K (02))
Program Officer
Nasseri, M Faraz
Project Start
1992-04-01
Project End
2012-05-31
Budget Start
2011-06-01
Budget End
2012-05-31
Support Year
20
Fiscal Year
2011
Total Cost
$318,250
Indirect Cost
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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