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Znc2 module of RAG1 contributes towards structure-specific nuclease activity of RAGs

Nilavar, NM and Nishana, M and Paranjape, AM and Mahadeva, R and Kumari, R and Choudhary, B and Raghavan, SC (2020) Znc2 module of RAG1 contributes towards structure-specific nuclease activity of RAGs. In: Biochemical Journal, 477 (18). pp. 3567-3582.

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Official URL: https://doi.org/10.1042/BCJ20200361


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. © 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society

Item Type: Journal Article
Publication: Biochemical Journal
Publisher: Portland Press Ltd
Additional Information: The copyright for this article belongs to the Author(s).
Keywords: amino acid; heteroduplex; nuclease; RAG1 protein; RAG2 protein; ribonuclease H; single stranded DNA; DNA binding protein; heteroduplex; homeodomain protein; nuclear protein; RAG-1 protein; RAG2 protein, human, Article; carboxy terminal sequence; catalyst; controlled study; DNA sequence; DNA structure; enzyme activity; gel mobility shift assay; gene deletion; human; point mutation; priority journal; protein domain; protein expression; protein purification; restriction site; site directed mutagenesis; structure activity relation; VDJ recombination; Western blotting; chemistry; genetics; HEK293 cell line; metabolism; protein motif, Amino Acid Motifs; DNA-Binding Proteins; HEK293 Cells; Homeodomain Proteins; Humans; Nuclear Proteins; Nucleic Acid Heteroduplexes; Protein Domains; Structure-Activity Relationship; V(D)J Recombination
Department/Centre: Division of Biological Sciences > Biochemistry
Date Deposited: 24 Jan 2023 04:25
Last Modified: 24 Jan 2023 04:25
URI: https://eprints.iisc.ac.in/id/eprint/79292

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