RAG transposase can capture and commit to target DNA before or after donorcleavage

The discovery that the V(D)J recombinase functions as a transposase in vitr o suggests that transposition by this system might he a potent source of ge nomic instability. To gain insight into the mechanisms that regulate transp osition, we investigated a phenomenon termed target commitment that reflect s a functional association between the RAG transposase and the target DNA. We found that the V(D)J recombinase is quite promiscuous, forming productiv e complexes with target DNA both before and after donor cleavage, and our d ata indicate that the rate-limiting step for transposition occurs after tar get capture. Formation of stable target capture complexes depends upon the presence of active-site metal binding residues (the DDE motif), suggesting that active-site amino acids in RAG-1 are critical for target capture. The ability of the RAG transposase to commit to target prior to cleavage may re sult in a preference for transposition into nearby targets, such as immunog lobulin and T-cell receptor loci. This could bias transposition toward rela tively "safe" regions of the genome. A preference for localized transpositi on may also have influenced the evolution of the antigen receptor loci.