Abstract The RAG1 and RAG2 proteins introduce double-strand DNA breaks at antigen-receptor loci in developing lymphocytes to initiate V(D)J recombination. How RAG proteins find the correct target locus in a vast excess of non-specific chromatin is not known. Here we measured dynamics of RAG1/RAG2 interactions with chromatin in living pro-B cells. We found that the majority of RAG1 or RAG1/RAG2 complex is in a fast 3D diffusive state, and the residual slow diffusive (bound) fraction was determined by a non-core portion of RAG1, and the PHD domain of RAG2. The RAG proteins exhibited distinct dynamics at the IgH locus. In particular, RAG2 increased the probability of RAG1 binding to IgH , a property that likely explains its non-catalytic role in V(D)J recombination. Our observations reveal how RAG finds its targets in developing B cells. One Sentence Summary Single-molecule imaging of the RAG recombinase reveals its search strategy for chromatin, H3K4me3 and antibody gene loci in living cells.

Direct observation of RAG recombinase recruitment to chromatin and the IgH locus in live pro-B cells