The DNA-bending protein, HMG1, is required for correct cleavage of 23 bp recombination signal sequences by recombination activating gene proteins in vitro.
DNA-bending proteins are known to facilitate the in vitro V(D)J joining of antigen receptor genes. Here we report that the high-mobility group protein, HMG1, is necessary for the correct nicking of the 23 bp recombination signal sequence (23-RSS) by the recombination [corrected] activating gene (RAG) proteins, RAG1 and RAG2. Without HMG1, the mouse Jkappa1 23-RSS was recognized as if it were the 12-RSS and nicked at a site 12 + 7 nucleotides away from the 9mer signal, even though no 7mer-like sequence was evident at the cryptic nicking site. When increased amounts of HMG1 were added, the 23-RSS substrate was nicked correctly at a site 23 + 7 nucleotides from the 9mer, and nicking at the cryptic site disappeared. Unlike the 23-RSS, the 12-RSS did not require HMG1 for correct nicking, although HMG1 was found to increase the interaction between RSS and RAG proteins. Modification-interference assays demonstrated that HMG1 caused changes in the interaction between the 23-RSS and RAG proteins specifically at the 7mer and the cryptic nicking site.
Yoshida
T
T
Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.
Tsuboi
A
A
Ishiguro
K i
Ki
Nagawa
F
F
Sakano
H
H
D000818:Animals; D004264:DNA Topoisomerases, Type I; D004268:DNA-Binding Proteins / Q000378:metabolism; D006609:High Mobility Group Proteins / Q000494:pharmacology*; D018398:Homeodomain Proteins / Q000378:metabolism*; D051379:Mice; D011506:Proteins
D004268:DNA-Binding Proteins; D006609:High Mobility Group Proteins; D018398:Homeodomain Proteins; D011506:Proteins; C104074:RSS protein, rat; C499308:Rag2 protein, mouse; C064655:V(D)J recombination activating protein 2; C064658:RAG-1 protein; D004264:DNA Topoisomerases, Type I