Znc2 module of RAG1 contributes towards structure-specific nuclease activity of RAGs.
Recombination activating genes (RAGs), consisting of RAG1 and RAG2 have ability to perform spatially and temporally regulated DNA recombination in a sequence specific manner. Besides, RAGs also cleave at non-B DNA structures and are thought to contribute towards genomic rearrangements and cancer. The nonamer binding domain of RAG1 binds to the nonamer sequence of the signal sequence during V(D)J recombination. However, deletion of NBD did not affect RAG cleavage on non-B DNA structures. In the present study, we investigated the involvement of other RAG domains when RAGs act as a structure-specific nuclease. Studies using purified central domain (CD) and C-terminal domain (CTD) of the RAG1 showed that CD of RAG1 exhibited high affinity and specific binding to heteroduplex DNA, which was irrespective of the sequence of single-stranded DNA, unlike CTD which showed minimal binding. Furthermore, we show that ZnC2 of RAG1 is crucial for its binding to DNA structures as deletion and point mutations abrogated the binding of CD to heteroduplex DNA. Our results also provide evidence that unlike RAG cleavage on RSS, central domain of RAG1 is sufficient to cleave heteroduplex DNA harbouring pyrimidines, but not purines. Finally, we show that a point mutation in the DDE catalytic motif is sufficient to block the cleavage of CD on heteroduplex DNA. Therefore, in the present study we demonstrate that the while ZnC2 module in central domain of RAG1 is required for binding to non-B DNA structures, active site amino acids are important for RAGs to function as a structure-specific nuclease.
['Nilavar|Namrata M|NM|', 'Nishana|Mayilaadumveettil|M|', 'Paranjape|Amita M|AM|', 'Mahadeva|Raghunandan|R|', 'Kumari|Rupa|R|', 'Choudhary|Bibha|B|', 'Raghavan|Sathees C|SC|']
D020816:Amino Acid Motifs D004268:DNA-Binding Proteins D057809:HEK293 Cells D018398:Homeodomain Proteins D006801:Humans D009687:Nuclear Proteins D009692:Nucleic Acid Heteroduplexes D000072417:Protein Domains D013329:Structure-Activity Relationship D060152:V(D)J Recombination
RAG2 non-B DNA structures V(D)J recombination RAG complex genomic integrity