title: Recruitment of RAG1 and RAG2 to Chromatinized DNA during V(D)J Recombination.
abstract: V(D)J recombination is initiated by the binding of the RAG1 and RAG2 proteins to recombination signal sequences (RSSs) that consist of conserved heptamer and nonamer sequences separated by a spacer of either 12 or 23 bp. Here, we used RAG-inducible pro-B v-Abl cell lines in conjunction with chromatin immunoprecipitation to better understand the protein and RSS requirements for RAG recruitment to chromatin. Using a catalytic mutant form of RAG1 to prevent recombination, we did not observe cooperation between RAG1 and RAG2 in their recruitment to endogenous Jκ gene segments over a 48-h time course. Using retroviral recombination substrates, we found that RAG1 was recruited inefficiently to substrates lacking an RSS or containing a single RSS, better to substrates with two 12-bp RSSs (12RSSs) or two 23-bp RSSs (23RSSs), and more efficiently to a substrate with a 12/23RSS pair. RSS mutagenesis demonstrated a major role for the nonamer element in RAG1 binding, and correspondingly, a cryptic RSS consisting of a repeat of CA dinucleotides, which poorly re-creates the nonamer, was ineffective in recruiting RAG1. Our findings suggest that 12RSS-23RSS cooperation (the "12/23 rule") is important not only for regulating RAG-mediated DNA cleavage but also for the efficiency of RAG recruitment to chromatin.
authors: Department of Immunobiology, Yale University School of Medicine, New Haven, Connecticut, USA.
authors: Keerthi
authors: K
authors: Shetty
authors: Department of Immunobiology, Yale University School of Medicine, New Haven, Connecticut, USA Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut, USA david.schatz@yale.edu.
authors: David G
authors: DG
authors: Schatz
mesh_terms: D000818:Animals; D002460:Cell Line; D002843:Chromatin / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D053837:DNA Cleavage; D004268:DNA-Binding Proteins / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000378:metabolism*; D051379:Mice; D011485:Protein Binding; D060152:V(D)J Recombination
chemical_list: D002843:Chromatin; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C064655:V(D)J recombination activating protein 2; C064658:RAG-1 protein; D004247:DNA