Full-length RAG1 promotes contact with coding and intersignal sequences in RAG protein complexes bound to recombination signals paired in cis.
The RAG proteins initiate V(D)J recombination by mediating synapsis and cleavage of two different antigen receptor gene segments through interactions with their flanking recombination signal sequences (RSS). The protein-DNA complexes that support this process have mainly been studied using RAG-RSS complexes assembled using oligonucleotide substrates containing a single RSS that are paired in trans to promote synapsis. How closely these complexes model those formed on longer, more physiologically relevant substrates containing RSSs on the same DNA molecule (in cis) remains unclear. To address this issue, we characterized discrete core and full-length RAG protein complexes bound to RSSs paired in cis. We find these complexes support cleavage activity regulated by V(D)J recombination's '12/23 rule' and exhibit plasticity in RSS usage dependent on partner RSS composition. DNA footprinting studies suggest that the RAG proteins in these complexes mediate more extensive contact with sequences flanking the RSS than previously observed, some of which are enhanced by full-length RAG1, and associated with synapsis and efficient RSS cleavage. Finally, we demonstrate that the RAG1 C-terminus facilitates hairpin formation on long DNA substrates, and full-length RAG1 promotes hairpin retention in the post-cleavage RAG complex. These results provide new insights into the mechanism of physiological V(D)J recombination.
Kumar
Sushil
S
Department of Medical Microbiology and Immunology, Creighton University Medical Center, Omaha, Nebraska 68178, USA.
Swanson
Patrick C
PC
D001665:Binding Sites; D020134:Catalytic Domain; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D018983:DNA Footprinting; D015321:Gene Rearrangement; D024243:HMGB1 Protein / Q000378:metabolism; D018398:Homeodomain Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008745:Methylation; D009154:Mutation; D011485:Protein Binding; D011995:Recombination, Genetic; D012045:Regulatory Sequences, Nucleic Acid
D024243:HMGB1 Protein; D018398:Homeodomain Proteins; C064658:RAG-1 protein; D004247:DNA