A C-terminal region of RAG1 contacts the coding DNA during V(D)J recombination.
The site-specific DNA rearrangement process, called V(D)J recombination, creates much of the diversity of immune receptor molecules in the adaptive immune system. Central to this reaction is the organization of the protein-DNA complex containing the proteins RAG1 and RAG2 and their DNA targets. A long-term goal is to appreciate the three-dimensional relationships between the proteins and DNA that allow the assembly of the appropriate reaction intermediates, resulting in concerted cleavage and directed rejoining of the DNA ends. Previous cross-linking approaches have mapped RAG1 contacts on the DNA. RAG1 protein contacts the DNA at the conserved heptamer and nonamer sequences as well as at the coding DNA adjacent to the heptamer. Here we subject RAG1, covalently cross-linked to DNA substrates, to partial cyanogen bromide degradation or trypsin proteolysis in order to map contacts on the protein. We find that coding-sequence contacts occur near the C terminus of RAG1, while contacts made within the recombination signal sequence occur nearer the N terminus of the core region of RAG1. A deletion protein lacking the C-terminal DNA-contacting region is still capable of making the N-terminal contacts. This suggests that the two binding interactions may exist on two separate domains of the protein. A trypsin cleavage pattern of the native protein supports this conclusion. A two-domain model for RAG1 is evaluated with respect to the larger recombination complex.
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Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia 30912, USA.
Bailin
T
T
Sadofsky
M J
MJ
D001483:Base Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents; D003488:Cyanogen Bromide / Q000737:chemistry / Q000378:metabolism; D015342:DNA Probes / Q000737:chemistry / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000379:methods; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism
D003432:Cross-Linking Reagents; D015342:DNA Probes; D018398:Homeodomain Proteins; D011994:Recombinant Proteins; C064658:RAG-1 protein; D014357:Trypsin; D003488:Cyanogen Bromide