V(D)J recombination: modulation of RAG1 and RAG2 cleavage activity on 12/23 substrates by whole cell extract and DNA-bending proteins.
Antigen receptor gene rearrangement is directed by DNA motifs consisting of a conserved heptamer and nonamer separated by a nonconserved spacer of either 12 or 23 base pairs (12 or 23 recombination signal sequences [RSS]). V(D)J recombination requires that the rearranging DNA segments be flanked by RSSs of different spacer lengths, a phenomenon known as the 12/23 rule. Recent studies have shown that this restriction operates at the level of DNA cleavage, which is mediated by the products of the recombination activating genes RAG1 and RAG2. Here, we show that RAG1 and RAG2 are not sufficient for 12/23 dependent cleavage, whereas RAG1 and RAG2 complemented with whole cell extract faithfully recapitulates the 12/23 rule. In addition, HMG box containing proteins HMG1 and HMG2 enhance RAG1- and RAG2-mediated cleavage of substrates containing 23 RSS but not of substrates containing only 12 RSS. These results suggest the existence of a nucleoprotein complex at the cleavage site, consisting of architectural, catalytic, and regulatory components.
Sawchuk
D J
DJ
Laboratory of Molecular Immunology, The Rockefeller University, New York 10021, USA.
Weis-Garcia
F
F
Malik
S
S
Besmer
E
E
Bustin
M
M
Nussenzweig
M C
MC
Cortes
P
P
D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004254:DNA Nucleotidyltransferases / Q000378:metabolism; D004268:DNA-Binding Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D006609:High Mobility Group Proteins / Q000378:metabolism; D018398:Homeodomain Proteins; D006801:Humans; D009687:Nuclear Proteins; D009698:Nucleoproteins / Q000378:metabolism*; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011948:Receptors, Antigen, T-Cell / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic / Q000235:genetics*; D013601:T-Lymphocytes / Q000378:metabolism*; D014162:Transfection / Q000235:genetics; D045567:VDJ Recombinases
D004268:DNA-Binding Proteins; D006609:High Mobility Group Proteins; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; D009698:Nucleoproteins; D011506:Proteins; C499311:RAG2 protein, human; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins; C064655:V(D)J recombination activating protein 2; C064658:RAG-1 protein; D004247:DNA; D004254:DNA Nucleotidyltransferases; D045567:VDJ Recombinases