Abstract
RAG complex consisting of RAG1 and RAG2 is a site-specific endonuclease responsible for the generation of antigen receptor diversity. It cleaves recombination signal sequence (RSS), comprising of conserved heptamer and nonamer. Nonamer binding domain (NBD) of RAG1 plays a central role in the recognition of RSS. To investigate the DNA binding properties of the domain, NBD of murine RAG1 was cloned, expressed and purified. Electrophoretic mobility shift assays showed that NBD binds with high affinity to nonamer in the context of 12/23 RSS or heteroduplex DNA. NBD binding was specific to thymines when single stranded DNA containing poly A, C, G or T were used. Biolayer interferometry studies showed that poly T binding to NBD was robust and comparable to that of 12RSS. More than 23 nt was essential for NBD binding at homothymidine stretches. On a double-stranded DNA, NBD could bind to A:T stretches, but not G:C or random sequences. Although NBD is indispensable for sequence specific activity of RAGs, external supplementation of purified nonamer binding domain to NBD deleted cRAG1/cRAG2 did not restore its activity, suggesting that the overall domain architecture of RAG1 is important. Therefore, we define the sequence requirements of NBD binding to DNA.
Highlights
IntroductionRAGs (recombination activating genes), the nuclease responsible for V(D)J recombination consists of RAG1 and RAG21,2
RAGs, the nuclease responsible for V(D)J recombination consists of RAG1 and RAG21,2
The nonamer binding region corresponding to amino acids 384–461 were PCR amplified from RAG1 gene
Summary
RAGs (recombination activating genes), the nuclease responsible for V(D)J recombination consists of RAG1 and RAG21,2. Besides its role in V(D)J recombination as a sequence-specific nuclease, RAGs can act as a structure-specific nuclease wherein it recognizes altered DNA structures and introduces nick at double strand (ds)-single strand (ss) transition. Recent studies have revealed that the structure-specific activity of RAGs is regulated by sequence of both the single-stranded DNA and flanking double-stranded DNA19,20. NBD at the N-terminus of cRAG1, recognizes and binds to nonamer of RSS and aids in anchoring RAGs onto RSS, while other domains are responsible for its catalytic activity and its interaction with RAG225,26. Since a nonamer adjacent to a non-B DNA structure could facilitate its cleavage, characterizing the role of NBD in structure-specific activity of RAGs is of significance. We define the sequence requirements of NBD binding to double- and single-stranded DNA
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