This is a competing renewal application requesting five years of support to continue the applicant's research program on the regulation and function of the RAG1 and RAG2 proteins during V(D)J recombination. During the previous funding period, collaborative experiments between the laboratories of the applicant and Gellert demonstrated that site specific cleavage at a recombination signal sequence (RSS) can be recapitulated in vitro with purified RAG1 and RAG2. In particular, these experiments showed that RAG1 and RAG2 are both necessary and sufficient for the site specific cleavage reaction, which generates a coding end with a hairpin structure and a blunt 5'-phosphorylated signal end. The applicant now proposes to use the in vitro V(D)J recombination assay to ask a number of very specific questions regarding the biochemical functions of the RAG1 and RAG2 proteins in the V(D)J cleavage reaction. Four distinct but interrelated specific aims are presented in this application. An RSS consists of a dyad-symmetric heptamer sequence that is separated from an A/T rich nonomer by a spacer region of nonconserved nucleotides. The spacer region is either 12 or 23 nucleotides in length. A defining feature of the V(D)J cleavage reaction in vivo is that it requires two RSSs, one with a 12 nt spacer and the other with 23nt spacer, the 12/23 rule. It is believed that these two distinct RSSs are brought together in a synaptic complex to effect a coupled cleavage. The objective of the first aim is to dissect the mechanism of coupled cleavage. In vitro experiments are proposed to determine the DNA sequence and structure requirements for the assembly of a 12/23 RSS pair in a RAG complex that mediates coupled cleavage. The objective of the second aim is to identify and biochemically characterize functional complexes between the RAG proteins and RSS DNA sequences. In particular RAG/DNA complexes formed on single 12 or 23 RSSs will be compared to those formed on 12/23 RSS pairs. A mutational analysis of the RAG1 and RAG2 proteins will be conducted in the third aim in order to identify DNA binding domains, the active site for cleavage, and regions involved in forming higher order structures.
The fourth aim i nvestigates the role of the RAG proteins in the regulation of V(D)J recombination.
This aim builds on the applicant's observation that a C-terminal truncation of RAG2 affects the efficiency of V-to-DJ joining but not that of D-to-J joining, an observation suggesting that the RAG proteins themselves may be involved in ordering rearrangements at the V-D-J loci. Particular RAG mutants will be expressed in cultured lymphocytes and transgenic mice to ask if they perturb the lineage- and stage-specific control of V(D)J recombination. In addition, the yeast two-hybrid assay will be employed to screen for potential regulatory factors that interact with the C-terminal domain of RAG2.
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