The central domain of core RAG1 preferentially recognizes single-stranded recombination signal sequence heptamer.
RAG1 and RAG2 initiate V(D)J recombination by introducing DNA double strand breaks between each selected gene segment and its bordering recombination signal sequence (RSS) in a two-step mechanism in which the DNA is first nicked, followed by hairpin formation. The RSS consists of a conserved nonamer and heptamer sequence, in which the latter borders the site of DNA cleavage. A region within RAG1, referred to as the central domain (residues 528-760 of 1040 in the full-length protein), has been shown previously to bind specifically to the double-stranded (ds) RSS heptamer, but with both weak specificity and affinity. However, additional investigations into the RAG1-RSS heptamer interaction are required because the DNA substrate forms intermediate conformations during the V(D)J recombination reaction. These include the nicked and hairpin products, as well as likely base unpairing to produce single-stranded (ss) DNA near the cleavage site. Here, it was determined that although the central domain showed substantially higher binding affinity for ss and nicked versus ds substrate, the interaction with ss RSS was particularly robust. In addition, the central domain bound with greater sequence specificity to the ss RSS heptamer than to the ds form. This study provides important insight into the V(D)J recombination reaction, specifically that significant interaction of the RSS heptamer with RAG1 occurs only after the induction of conformational changes at the RSS heptamer.
Peak
Mandy M
MM
Department of Biochemistry and Molecular Biology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190, USA.
Arbuckle
Janeen L
JL
Rodgers
Karla K
KK
D000818:Animals; D001665:Binding Sites; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004254:DNA Nucleotidyltransferases / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005982:Glutathione Transferase / Q000378:metabolism; D018398:Homeodomain Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D009841:Oligonucleotides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011995:Recombination, Genetic; D045567:VDJ Recombinases
D004277:DNA, Single-Stranded; D018398:Homeodomain Proteins; D009841:Oligonucleotides; C064658:RAG-1 protein; D004247:DNA; D005982:Glutathione Transferase; D004254:DNA Nucleotidyltransferases; D045567:VDJ Recombinases