The vast antigen receptor repertoire of the adaptive immune system is generated through recombination of the (V)ariable, (D)iversity, and (J)oining gene segments. This process, aptly termed V(D)J recombination, is mediated by the protein products of the recombination activating genes, Rag1 and Rag2, in three steps: recognition/synapsis, cleavage, and joining. The first two steps are relatively well understood; RAG1 and RAG2 act in concert as the endonuclease that cleaves precisely between conserved recombination signal sequences (RSS) and flanking V, D, or J coding sequences producing two distinct intermediates, blunt signal ends and covalently sealed hairpin coding ends. The subsequent joining phase of these RAG mediated double strand breaks (DSBs) is far less characterized and involves precisely ligating the signal ends to each other, while the hairpin coding ends are opened and joined in a reaction that results in nucleotide loss or addition. At least three pathways exist to protect the genome by repairing DSBs: homologous recombination (HR), classical nonhomologous end joining (NHEJ), and a poorly defined, less accurate joining pathway referred to as alternative NHEJ. Although classical NHEJ factors and the RAG proteins have been genetically implicated in V(D)J joining, the mechanism of DNA repair pathway selection has yet to be elucidated.
My thesis work focuses on the joining phase of V(D)J recombination, specifically how the RAG complex influences DNA repair pathway selection. In collaboration with other lab members, I identified a C-terminal frameshift mutation in RAG2 that retains recombination activity while allowing the V(D)J intermediates to be abnormally accessed by alternative NHEJ and HR pathways. Of particular interest, this mutation lands within an unexamined acidic region of RAG2, which I further characterized by genetic analysis to be essential in restricting the end joining of V(D)J intermediates to classical NHEJ. Through biochemical analysis, I also identified mutations within this acidic region that destabilize the RAG-mediated post cleavage complex (PCC), supporting the hypothesis that alternative end processing occurs when the V(D)J intermediates are not properly shepherded to classical NHEJ by the RAG proteins. These studies have important implications for the mechanism of joining and suggest a role for RAG proteins in maintenance of genomic stability in developing lymphocytes.
|Advisor:||Roth, David B.|
|Commitee:||Dustin, Michael L., Lafaille, Juan J., Smith, Susan L., Zhu, Chengming (Ben)|
|School:||New York University|
|Department:||Basic Medical Science|
|School Location:||United States -- New York|
|Source:||DAI-B 70/07, Dissertation Abstracts International|
|Subjects:||Molecular biology, Immunology|
|Keywords:||Acidic region, DNA repair, Non-homologous end joining, RAG2, V(D)J recombination|
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