{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T15:37:53Z","timestamp":1790264273892,"version":"4.1.0"},"reference-count":50,"publisher":"Portland Press Ltd.","issue":"18","license":[{"start":{"date-parts":[[2020,9,4]],"date-time":"2020-09-04T00:00:00Z","timestamp":1599177600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/2.0\/"}],"content-domain":{"domain":["portlandpress.com"],"crossmark-restriction":true},"short-container-title":[],"published-print":{"date-parts":[[2020,9,30]]},"abstract":"<jats:p>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.<\/jats:p>","DOI":"10.1042\/bcj20200361","type":"journal-article","created":{"date-parts":[[2020,9,4]],"date-time":"2020-09-04T13:23:28Z","timestamp":1599225808000},"page":"3567-3582","update-policy":"https:\/\/doi.org\/10.1042\/crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Znc2 module of RAG1 contributes towards structure-specific nuclease activity of RAGs"],"prefix":"10.1042","volume":"477","author":[{"given":"Namrata M.","family":"Nilavar","sequence":"first","affiliation":[{"name":"Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mayilaadumveettil","family":"Nishana","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Amita M.","family":"Paranjape","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Raghunandan","family":"Mahadeva","sequence":"additional","affiliation":[{"name":"Institute of Bioinformatics and Applied Biotechnology, Electronics City, Bangalore 560100, India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rupa","family":"Kumari","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bibha","family":"Choudhary","sequence":"additional","affiliation":[{"name":"Institute of Bioinformatics and Applied Biotechnology, Electronics City, Bangalore 560100, India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3003-1417","authenticated-orcid":false,"given":"Sathees C.","family":"Raghavan","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"288","published-online":{"date-parts":[[2020,9,24]]},"reference":[{"key":"2021112218131142200_BCJ-2020-0361C1","doi-asserted-by":"publisher","first-page":"23","DOI":"10.1385\/IR:23:1:23","article-title":"RAG1 and RAG2 in V(D)J recombination and transposition","volume":"23","year":"2001","journal-title":"Immunol. Res."},{"key":"2021112218131142200_BCJ-2020-0361C2","doi-asserted-by":"publisher","first-page":"359","DOI":"10.1146\/annurev.iy.10.040192.002043","article-title":"V(D)J recombination: molecular biology and regulation","volume":"10","year":"1992","journal-title":"Annu. Rev. Immunol."},{"key":"2021112218131142200_BCJ-2020-0361C3","doi-asserted-by":"publisher","first-page":"167","DOI":"10.1146\/annurev-genet-110410-132552","article-title":"V(D)J recombination: mechanisms of initiation","volume":"45","year":"2011","journal-title":"Annu. Rev. Genet."},{"key":"2021112218131142200_BCJ-2020-0361C4","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1016\/bs.ai.2015.07.002","article-title":"Regulation and evolution of the RAG recombinase","volume":"128","year":"2015","journal-title":"Adv. Immunol."},{"key":"2021112218131142200_BCJ-2020-0361C5","doi-asserted-by":"publisher","first-page":"189","DOI":"10.1006\/smim.1997.0070","article-title":"The end-joining reaction in V(D)J recombination","volume":"9","year":"1997","journal-title":"Semin. Immunol."},{"key":"2021112218131142200_BCJ-2020-0361C6","doi-asserted-by":"publisher","first-page":"701","DOI":"10.1002\/1521-4141(200203)32:3 &lt;701::AID-IMMU701&gt;3.0.CO;2-T","article-title":"Different types of V(D)J recombination and end-joining defects in DNA double-strand break repair mutant mammalian cells","volume":"32","year":"2002","journal-title":"Eur. J. Immunol."},{"key":"2021112218131142200_BCJ-2020-0361C7","doi-asserted-by":"publisher","first-page":"233","DOI":"10.1007\/s12026-012-8329-z","article-title":"Role of non-homologous end joining in V(D)J recombination","volume":"54","year":"2012","journal-title":"Immunol. Res."},{"key":"2021112218131142200_BCJ-2020-0361C8","doi-asserted-by":"publisher","first-page":"88","DOI":"10.1038\/nature02355","article-title":"A non-B-DNA structure at the Bcl-2 major breakpoint region is cleaved by the RAG complex","volume":"428","year":"2004","journal-title":"Nature"},{"key":"2021112218131142200_BCJ-2020-0361C9","doi-asserted-by":"publisher","first-page":"278","DOI":"10.1016\/j.dnarep.2005.09.008","article-title":"Hybrid joint formation in human V(D)J recombination requires nonhomologous DNA end joining","volume":"5","year":"2006","journal-title":"DNA Repair (Amst)"},{"key":"2021112218131142200_BCJ-2020-0361C10","doi-asserted-by":"publisher","first-page":"115","DOI":"10.1042\/BJ20121031","article-title":"A non-B DNA can replace heptamer of V(D)J recombination when present along with a nonamer: implications in chromosomal translocations and cancer","volume":"448","year":"2012","journal-title":"Biochem. J."},{"key":"2021112218131142200_BCJ-2020-0361C11","doi-asserted-by":"publisher","first-page":"7587","DOI":"10.1074\/jbc.M109.089631","article-title":"Cytosines, but not purines, determine recombination activating gene (RAG)-induced breaks on heteroduplex DNA structures: implications for genomic instability","volume":"285","year":"2010","journal-title":"J. Biol. Chem."},{"key":"2021112218131142200_BCJ-2020-0361C12","doi-asserted-by":"publisher","first-page":"6180","DOI":"10.1093\/nar\/gkg819","article-title":"DNA mismatches and GC-rich motifs target transposition by the RAG1\/RAG2 transposase","volume":"31","year":"2003","journal-title":"Nucleic Acids Res."},{"key":"2021112218131142200_BCJ-2020-0361C13","doi-asserted-by":"publisher","first-page":"4","DOI":"10.1111\/febs.13121","article-title":"Structure-specific nuclease activity of RAGs is modulated by sequence, length and phase position of flanking double-stranded DNA","volume":"282","year":"2015","journal-title":"FEBS J."},{"key":"2021112218131142200_BCJ-2020-0361C14","doi-asserted-by":"publisher","first-page":"1439","DOI":"10.1084\/jem.20030627","article-title":"Impaired V(D)J recombination and lymphocyte development in core RAG1-expressing mice","volume":"198","year":"2003","journal-title":"J. Exp. Med."},{"key":"2021112218131142200_BCJ-2020-0361C15","doi-asserted-by":"publisher","first-page":"507","DOI":"10.1038\/nature14174","article-title":"Crystal structure of the V(D)J recombinase RAG1-RAG2","volume":"518","year":"2015","journal-title":"Nature"},{"key":"2021112218131142200_BCJ-2020-0361C16","doi-asserted-by":"publisher","first-page":"72","DOI":"10.1016\/j.tibs.2016.10.003","article-title":"Riches in RAGs: revealing the V(D)J recombinase through high-resolution structures","volume":"42","year":"2017","journal-title":"Trends Biochem. Sci."},{"key":"2021112218131142200_BCJ-2020-0361C17","doi-asserted-by":"publisher","first-page":"358","DOI":"10.1016\/j.molcel.2018.03.008","article-title":"Cracking the DNA code for V(D)J recombination","volume":"70","year":"2018","journal-title":"Mol. Cell"},{"key":"2021112218131142200_BCJ-2020-0361C18","doi-asserted-by":"publisher","first-page":"253","DOI":"10.1016\/S0092-8674(00)81343-4","article-title":"RAG1 mediates signal sequence recognition and recruitment of RAG2 in V(D)J recombination","volume":"87","year":"1996","journal-title":"Cell"},{"key":"2021112218131142200_BCJ-2020-0361C19","doi-asserted-by":"publisher","first-page":"935","DOI":"10.1016\/S1097-2765(00)80223-3","article-title":"The RAG1\/RAG2 complex constitutes a 3\u2032 flap endonuclease: implications for junctional diversity in V(D)J and transpositional recombination","volume":"4","year":"1999","journal-title":"Mol. Cell"},{"key":"2021112218131142200_BCJ-2020-0361C20","doi-asserted-by":"publisher","first-page":"475","DOI":"10.1016\/j.jmb.2011.11.002","article-title":"Differential reaction kinetics, cleavage complex formation, and nonamer binding domain dependence dictate the structure-specific and sequence-specific nuclease activity of RAGs","volume":"415","year":"2012","journal-title":"J. Mol. Biol."},{"key":"2021112218131142200_BCJ-2020-0361C21","doi-asserted-by":"publisher","first-page":"6850","DOI":"10.1128\/MCB.24.15.6850-6860.2004","article-title":"DNA cleavage activity of the V(D)J recombination protein RAG1 is autoregulated","volume":"24","year":"2004","journal-title":"Mol. Cell. Biol."},{"key":"2021112218131142200_BCJ-2020-0361C22","doi-asserted-by":"publisher","first-page":"37093","DOI":"10.1074\/jbc.M105988200","article-title":"Identification of two topologically independent domains in RAG1 and their role in macromolecular interactions relevant to V(D)J recombination","volume":"276","year":"2001","journal-title":"J. Biol. Chem."},{"key":"2021112218131142200_BCJ-2020-0361C23","doi-asserted-by":"publisher","first-page":"936","DOI":"10.1093\/nar\/gkq824","article-title":"Formation of a G-quadruplex at the BCL2 major breakpoint region of the t(14;18) translocation in follicular lymphoma","volume":"39","year":"2011","journal-title":"Nucleic Acids Res."},{"key":"2021112218131142200_BCJ-2020-0361C24","doi-asserted-by":"publisher","first-page":"288","DOI":"10.1016\/j.isci.2019.10.033","article-title":"G-quadruplex structures contribute to differential radiosensitivity of the human genome","volume":"21","year":"2019","journal-title":"iScience"},{"key":"2021112218131142200_BCJ-2020-0361C25","doi-asserted-by":"publisher","first-page":"924","DOI":"10.1038\/nprot.2007.132","article-title":"Gene splicing and mutagenesis by PCR-driven overlap extension","volume":"2","year":"2007","journal-title":"Nat. Protoc."},{"key":"2021112218131142200_BCJ-2020-0361C26","doi-asserted-by":"publisher","first-page":"e2852","DOI":"10.1038\/cddis.2017.237","article-title":"HIV integrase inhibitor, elvitegravir, impairs RAG functions and inhibits V(D)J recombination","volume":"8","year":"2017","journal-title":"Cell Death Dis."},{"key":"2021112218131142200_BCJ-2020-0361C27","doi-asserted-by":"publisher","first-page":"2938","DOI":"10.1093\/nar\/27.14.2938","article-title":"A dimer of the lymphoid protein RAG1 recognizes the recombination signal sequence and the complex stably incorporates the high mobility group protein HMG2","volume":"27","year":"1999","journal-title":".Nucleic Acids Res."},{"key":"2021112218131142200_BCJ-2020-0361C28","doi-asserted-by":"publisher","first-page":"8688","DOI":"10.1074\/jbc.M111.307363","article-title":"Mechanism of fragility at BCL2 gene minor breakpoint cluster region during t(14;18) chromosomal translocation","volume":"287","year":"2012","journal-title":"J. Biol. Chem."},{"key":"2021112218131142200_BCJ-2020-0361C29","doi-asserted-by":"publisher","first-page":"50","DOI":"10.1038\/s41420-020-0281-4","article-title":"Biochemical activity of RAGs is impeded by Dolutegravir, an HIV integrase inhibitor","volume":"6","year":"2020","journal-title":"Cell Death Discov."},{"key":"2021112218131142200_BCJ-2020-0361C30","doi-asserted-by":"publisher","first-page":"197","DOI":"10.1016\/j.jmb.2012.01.029","article-title":"Time-dependent predominance of nonhomologous DNA end-joining pathways during embryonic development in mice","volume":"417","year":"2012","journal-title":"J. Mol. Biol."},{"key":"2021112218131142200_BCJ-2020-0361C31","doi-asserted-by":"publisher","first-page":"4670","DOI":"10.1128\/MCB.18.8.4670","article-title":"Distinct roles of RAG1 and RAG2 in binding the V(D)J recombination signal sequences","volume":"18","year":"1998","journal-title":"Mol. Cell. Biol."},{"key":"2021112218131142200_BCJ-2020-0361C32","doi-asserted-by":"publisher","first-page":"19091","DOI":"10.1038\/srep19091","article-title":"Biochemical characterization of nonamer binding domain of RAG1 reveals its thymine preference with respect to length and position","volume":"6","year":"2016","journal-title":"Sci. Rep."},{"key":"2021112218131142200_BCJ-2020-0361C33","doi-asserted-by":"publisher","first-page":"1474","DOI":"10.1016\/j.cell.2012.11.054","article-title":"An inhibitor of nonhomologous end-joining abrogates double-strand break repair and impedes cancer progression","volume":"151","year":"2012","journal-title":"Cell"},{"key":"2021112218131142200_BCJ-2020-0361C34","doi-asserted-by":"publisher","first-page":"e1697","DOI":"10.1038\/cddis.2015.58","article-title":"Homology and enzymatic requirements of microhomology-dependent alternative end joining","volume":"6","year":"2015","journal-title":"Cell Death Dis."},{"key":"2021112218131142200_BCJ-2020-0361C35","volume-title":"Davidson's The Biochemistry of the Nucleic Acids","year":"1976","edition":"8th edn"},{"key":"2021112218131142200_BCJ-2020-0361C36","doi-asserted-by":"publisher","first-page":"720","DOI":"10.1016\/j.jmb.2015.09.014","article-title":"The HADDOCK2.2 Web server: user-friendly integrative modeling of biomolecular complexes","volume":"428","year":"2016","journal-title":"J. Mol. Biol."},{"key":"2021112218131142200_BCJ-2020-0361C37","doi-asserted-by":"publisher","first-page":"1605","DOI":"10.1002\/jcc.20084","article-title":"UCSF chimera\u2013a visualization system for exploratory research and analysis","volume":"25","year":"2004","journal-title":"J. Comput. Chem."},{"key":"2021112218131142200_BCJ-2020-0361C38","doi-asserted-by":"publisher","first-page":"18235","DOI":"10.1074\/jbc.M302041200","article-title":"The central domain of core RAG1 preferentially recognizes single-stranded recombination signal sequence heptamer","volume":"278","year":"2003","journal-title":"J. Biol. Chem."},{"key":"2021112218131142200_BCJ-2020-0361C39","doi-asserted-by":"publisher","first-page":"14008","DOI":"10.1073\/pnas.93.24.14008","article-title":"In vitro V(D)J recombination: signal joint formation","volume":"93","year":"1996","journal-title":"Proc. Natl Acad. Sci. U.S.A."},{"key":"2021112218131142200_BCJ-2020-0361C40","doi-asserted-by":"publisher","first-page":"1934","DOI":"10.1101\/gad.984502","article-title":"Evidence of a critical architectural function for the RAG proteins in end processing, protection, and joining in V(D)J recombination","volume":"16","year":"2002","journal-title":"Genes Dev."},{"key":"2021112218131142200_BCJ-2020-0361C41","doi-asserted-by":"publisher","first-page":"70","DOI":"10.1006\/jmbi.1996.0382","article-title":"A zinc-binding domain involved in the dimerization of RAG1","volume":"260","year":"1996","journal-title":"J. Mol. Biol."},{"key":"2021112218131142200_BCJ-2020-0361C42","doi-asserted-by":"publisher","first-page":"16325","DOI":"10.1074\/jbc.273.26.16325","article-title":"The effect of Me2+ cofactors at the initial stages of V(D)J recombination","volume":"273","year":"1998","journal-title":"J. Biol. Chem."},{"key":"2021112218131142200_BCJ-2020-0361C43","doi-asserted-by":"publisher","first-page":"449","DOI":"10.1128\/MCB.21.2.449-458.2001","article-title":"The DDE motif in RAG-1 is contributed in trans to a single active site that catalyzes the nicking and transesterification steps of V(D)J recombination","volume":"21","year":"2001","journal-title":"Mol. Cell. Biol."},{"key":"2021112218131142200_BCJ-2020-0361C44","doi-asserted-by":"publisher","first-page":"223","DOI":"10.1016\/S0092-8674(00)80977-0","article-title":"The three chemical steps of Tn10\/IS10 transposition involve repeated utilization of a single active site","volume":"84","year":"1996","journal-title":"Cell"},{"key":"2021112218131142200_BCJ-2020-0361C45","doi-asserted-by":"publisher","first-page":"499","DOI":"10.1038\/nsmb.1593","article-title":"Structure of the RAG1 nonamer binding domain with DNA reveals a dimer that mediates DNA synapsis","volume":"16","year":"2009","journal-title":"Nat. Struct. Mol. Biol."},{"key":"2021112218131142200_BCJ-2020-0361C46","doi-asserted-by":"publisher","first-page":"3460","DOI":"10.1128\/MCB.22.10.3460-3473.2002","article-title":"Mutational analysis of all conserved basic amino acids in RAG-1 reveals catalytic, step arrest, and joining-deficient mutants in the V(D)J recombinase","volume":"22","year":"2002","journal-title":"Mol. Cell. Biol."},{"key":"2021112218131142200_BCJ-2020-0361C47","doi-asserted-by":"publisher","first-page":"2772","DOI":"10.1182\/blood.V97.9.2772","article-title":"Identical mutations in RAG1 or RAG2 genes leading to defective V(D)J recombinase activity can cause either T-B-severe combined immune deficiency or Omenn syndrome","volume":"97","year":"2001","journal-title":"Blood"},{"key":"2021112218131142200_BCJ-2020-0361C48","doi-asserted-by":"publisher","first-page":"885","DOI":"10.1016\/S0092-8674(00)81448-8","article-title":"Partial V(D)J recombination activity leads to Omenn syndrome","volume":"93","year":"1998","journal-title":"Cell"},{"key":"2021112218131142200_BCJ-2020-0361C49","doi-asserted-by":"publisher","first-page":"5683","DOI":"10.1128\/MCB.16.10.5683","article-title":"DNA sequence and structure requirements for cleavage of V(D)J recombination signal sequences","volume":"16","year":"1996","journal-title":"Mol. Cell. Biol."},{"key":"2021112218131142200_BCJ-2020-0361C50","doi-asserted-by":"publisher","first-page":"3197","DOI":"10.1002\/j.1460-2075.1996.tb00682.x","article-title":"Distinct DNA sequence and structure requirements for the two steps of V(D)J recombination signal cleavage","volume":"15","year":"1996","journal-title":"EMBO J."}],"container-title":["Biochemical Journal"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/portlandpress.com\/biochemj\/article-pdf\/477\/18\/3567\/893782\/bcj-2020-0361.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/portlandpress.com\/biochemj\/article-pdf\/477\/18\/3567\/893782\/bcj-2020-0361.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2021,11,23]],"date-time":"2021-11-23T02:31:48Z","timestamp":1637634708000},"score":1,"resource":{"primary":{"URL":"https:\/\/portlandpress.com\/biochemj\/article\/477\/18\/3567\/226329\/Znc2-module-of-RAG1-contributes-towards-structure"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,9,24]]},"references-count":50,"journal-issue":{"issue":"18","published-print":{"date-parts":[[2020,9,30]]}},"URL":"https:\/\/doi.org\/10.1042\/bcj20200361","relation":{},"ISSN":["0264-6021","1470-8728"],"issn-type":[{"value":"0264-6021","type":"print"},{"value":"1470-8728","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,9,24]]}}}