Footprint analysis of the RAG protein recombination signal sequence complex for V(D)J type recombination

被引:60
作者
Nagawa, F
Ishiguro, KI
Tsuboi, A
Yoshida, T
Ishikawa, A
Takemori, T
Otsuka, AJ
Sakano, H
机构
[1] Univ Tokyo, Grad Sch Sci, Dept Biochem & Biophys, Bunkyo Ku, Tokyo 113, Japan
[2] Natl Inst Infect Dis, Dept Immunol, Tokyo 162, Japan
[3] Illinois State Univ, Dept Biol Sci, Normal, IL 61790 USA
关键词
D O I
10.1128/MCB.18.1.655
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
We have studied the interaction between recombination signal sequences (RSSs) and protein products of the truncated forms of recombination-activating genes (RAG) by gel mobility shift, DNase I footprinting, and methylation interference assays. Methylation interference with dimethyl sulfate demonstrated that binding was blocked by methylation in the nonamer at the second-position G residue in the bottom strand and at the sixth- and seventh-position A residues in the top strand. DNase I footprinting experiments demonstrated that RAG1 alone, or even a RAG1 homeodomain peptide, gave footprint patterns very similar to those obtained with the RAG1-RAG2 complex. In the heptamer, partial methylation interference was observed at the sixth-position A residue in the bottom strand. In DNase I footprinting, the heptamer region was weakly protected in the bottom strand by RAG1. The effects of RSS mutations on RAG binding were evaluated by DNA footprinting. Comparison of the RAG-RSS footprint data with the published Hin model confirmed the notion that sequence-specific RSS RAG interaction takes place primarily between the Hin domain of the RAG1 protein and adjacent major and minor grooves of the nonamer DNA.
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页码:655 / 663
页数:9
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