Determinants of HMGB proteins required to promote RAG1/2-recombination signal sequence complex assembly and catalysis during V(D)J recombination.
Efficient assembly of RAG1/2-recombination signal sequence (RSS) DNA complexes that are competent for V(D)J cleavage requires the presence of the nonspecific DNA binding and bending protein HMGB1 or HMGB2. We find that either of the two minimal DNA binding domains of HMGB1 is effective in assembling RAG1/2-RSS complexes on naked DNA and stimulating V(D)J cleavage but that both domains are required for efficient activity when the RSS is incorporated into a nucleosome. The single-domain HMGB protein from Saccharomyces cerevisiae, Nhp6A, efficiently assembles RAG1/2 complexes on naked DNA; however, these complexes are minimally competent for V(D)J cleavage. Nhp6A forms much more stable DNA complexes than HMGB1, and a variety of mutations that destabilize Nhp6A binding to bent microcircular DNA promote increased V(D)J cleavage. One of the two DNA bending wedges on Nhp6A and the analogous phenylalanine wedge at the DNA exit site of HMGB1 domain A were found to be essential for promoting RAG1/2-RSS complex formation. Because the phenylalanine wedge is required for specific recognition of DNA kinks, we propose that HMGB proteins facilitate RAG1/2-RSS interactions by recognizing a distorted DNA structure induced by RAG1/2 binding. The resulting complex must be sufficiently dynamic to enable the series of RAG1/2-mediated chemical reactions on the DNA.
Dai
Yan
Y
Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114, USA.
Wong
Ben
B
Yen
Yi-Meng
YM
Oettinger
Marjorie A
MA
Kwon
Jongbum
J
Johnson
Reid C
RC
D000595:Amino Acid Sequence; D000818:Animals; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015321:Gene Rearrangement; D024243:HMGB1 Protein; D024001:HMGN Proteins; D006609:High Mobility Group Proteins / Q000235:genetics / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010649:Phenylalanine / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D045567:VDJ Recombinases / Q000235:genetics / Q000378:metabolism
D004268:DNA-Binding Proteins; D024243:HMGB1 Protein; D024001:HMGN Proteins; C104200:Hbp1 protein, rat; D006609:High Mobility Group Proteins; D018398:Homeodomain Proteins; C086471:NHP6A protein, S cerevisiae; D009687:Nuclear Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; C064655:V(D)J recombination activating protein 2; C064658:RAG-1 protein; D010649:Phenylalanine; D004247:DNA; D045567:VDJ Recombinases