title: Biochemical Characterization of Nonamer Binding Domain of RAG1 Reveals its Thymine Preference with Respect to Length and Position.
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.
authors.affiliation: Department of Biochemistry, Indian Institute of Science, Bangalore, 560 012, India.
authors.forename: Deepthi
authors.initials: D
authors.lastname: Raveendran
authors.affiliation: Department of Biochemistry, Indian Institute of Science, Bangalore, 560 012, India.
authors.forename: Sathees C
authors.initials: SC
authors.lastname: Raghavan
mesh_terms: D000818:Animals; D001402:B-Lymphocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D018398:Homeodomain Proteins / Q000737:chemistry* / Q000235:genetics; D051379:Mice; D011061:Poly A / Q000737:chemistry; D011066:Poly C / Q000737:chemistry; D011068:Poly G / Q000737:chemistry; D011071:Poly T / Q000737:chemistry*; D011485:Protein Binding; D000072417:Protein Domains; D021382:Protein Sorting Signals; D011946:Receptors, Antigen / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D060152:V(D)J Recombination
chemical_list: D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D021382:Protein Sorting Signals; C499308:Rag2 protein, mouse; D011946:Receptors, Antigen; D011994:Recombinant Proteins; C064658:RAG-1 protein; D011061:Poly A; D011071:Poly T; D011068:Poly G; D011066:Poly C