Characterization of the functional and structural properties of the V(D)J recombinase

The functional genes for the immunoglobulin and T cell receptor are assembled during development of B and T cells by a recombination mechanism known as V(D)J recombination. The initial steps are catalyzed by the recombination activating proteins RAG1 and RAG2, which generate DNA breaks at the site of recombination. Each RAG-mediated catalytic step in V(D)J recombination is dependent on the formation of a distinct assembly of the RAG proteins with the RSS.;In this study, we examined the association of RAG1, with and without RAG2, to consensus RSS versus non-RSS substrates. The results indicate that while RAG1 only can recognize the RSS, the sequence-specific interaction is masked by a high affinity non-sequence specific (NSS) DNA-binding mode. RAG1 forms a longer-lived complex with 12-RSS versus non sequence-specific DNA. Significantly, addition of RAG2 effectively suppressed the association of RAG1 with non-sequence specific DNA, resulting in a large differential in binding affinity for the RSS versus non-RSS sites.;We also obtained structural information on the complex containing the catalytically active core regions of RAG1 and RAG2. Using limited proteolysis combined with MALD-TOF mass spectrometry we found that RAG2 forms contacts with multiple regions of RAG1 including a major interaction site that bridges between the Central and C-terminal domains of core RAG1.;Finally, we have investigated subcellular localization of the RAG proteins and found that each RAG protein can influence cellular localization of the other RAG protein. Moreover, in response to DNA damage from ionizing radiation (IR), the RAG proteins were globally redistributed in the cell, with an apparent overall reduced accessibility to the genome.;Overall, our study provide new insight into the macromolecular assembly of the V(D)J recombinase and open a new avenue of investigation into a previously unknown mechanism for regulating the V(D)J recombinase during critical times in the cell.