Open AccessZnc2 module of RAG1 contributes towards structure-specific nuclease activity of RAGs
Author(s) -
Namrata M Nilavar,
Mayilaadumveettil Nishana,
Amita M Paranjape,
Raghunandan Mahadeva,
Richa Kumari,
Bibha Choudhary,
Sathees C. Raghavan
Publication year - 2020
Publication title -
biochemical journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.706
H-Index - 265
eISSN - 1470-8728
pISSN - 0264-6021
DOI - 10.1042/bcj20200361
Subject(s) - heteroduplex , nuclease , dna , recombination activating gene , biology , cleave , v(d)j recombination , rnase h , microbiology and biotechnology , cleavage (geology) , point mutation , gene , genetics , rnase p , mutation , recombination , rna , paleontology , fracture (geology)
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.