| First Author | Shetty K | Year | 2015 |
| Journal | Mol Cell Biol | Volume | 35 |
| Issue | 21 | Pages | 3701-13 |
| PubMed ID | 26303526 | Mgi Jnum | J:228215 |
| Mgi Id | MGI:5705677 | Doi | 10.1128/MCB.00219-15 |
| Citation | Shetty K, et al. (2015) Recruitment of RAG1 and RAG2 to Chromatinized DNA during V(D)J Recombination. Mol Cell Biol 35(21):3701-13 |
| abstractText | 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 Jkappa 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. |
