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Despite extensive molecular characterization, mechanisms and precise contributions of DNMT-specific methylation in native cellular contexts remain elusive. To address this, we employed structure-guided engineering of DNMT3A to enable catalytic transfer of extended azide tags from a synthetic cofactor analog, Ado-6-azide, onto DNA. By coupling orthogonal biocatalysis with pulse-internalization of Ado-6-azide or with metabolic conversion of the cofactor precursor, azido-methionine, we established two strategies for DNMT3A catalysis-dependent genome tagging in live mESCs under near-physiological conditions. Genome-wide mapping of DNMT3A catalysis (catalysome) in primed mouse embryonic stem cells facilitated discovery of opposing DNMT3A and TET dioxygenase activities at boundaries of CpG islands, promoter-proximal regions, and enhancers. DNMT3A also exhibited a strong enrichment at pericentric major satellites, reinforcing its key role in shaping the epigenetic centromere identity. Collectively with a complementary DNMT1-engineered cell line, we present a versatile platform for direct, selective, and time-resolved interrogation of individual DNA methylation writers in live cells under physiological conditions.<\/jats:p>","DOI":"10.1093\/nar\/gkag906","type":"journal-article","created":{"date-parts":[[2026,9,5]],"date-time":"2026-09-05T13:53:33Z","timestamp":1788616413000},"source":"Crossref","is-referenced-by-count":0,"title":["Live-cell DNMT3A \u201ccatalysome\u201d mapping using engineered methyl-transfer pathways"],"prefix":"10.1093","volume":"54","author":[{"given":"Vaidotas","family":"Stankevi\u010dius","sequence":"first","affiliation":[{"name":"Institute of Biotechnology, Life Sciences Center, Vilnius University , 10257 Vilnius ,","place":["Lithuania"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Liepa","family":"Gasiul\u0117","sequence":"additional","affiliation":[{"name":"Institute of Biotechnology, Life Sciences Center, Vilnius University , 10257 Vilnius ,","place":["Lithuania"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kotryna","family":"Kvederavi\u010di\u016bt\u0117","sequence":"additional","affiliation":[{"name":"Institute of Biotechnology, Life Sciences Center, Vilnius University , 10257 Vilnius ,","place":["Lithuania"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aleksandras","family":"\u010ce\u010dkauskas","sequence":"additional","affiliation":[{"name":"Institute of Biotechnology, Life Sciences Center, Vilnius University , 10257 Vilnius ,","place":["Lithuania"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Audron\u0117","family":"Ruk\u0161\u0117nait\u0117","sequence":"additional","affiliation":[{"name":"Institute of Biotechnology, Life Sciences Center, Vilnius University , 10257 Vilnius ,","place":["Lithuania"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Giedr\u0117","family":"Urbanavi\u010di\u016bt\u0117","sequence":"additional","affiliation":[{"name":"Institute of Biotechnology, Life Sciences Center, Vilnius University , 10257 Vilnius ,","place":["Lithuania"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gra\u017eina","family":"Petraityt\u0117","sequence":"additional","affiliation":[{"name":"Institute of Biotechnology, Life Sciences Center, Vilnius University , 10257 Vilnius ,","place":["Lithuania"]},{"name":"Institute of Chemistry, Faculty of Chemistry and Geosciences, Vilnius University , 03225 Vilnius ,","place":["Lithuania"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3974-597X","authenticated-orcid":false,"given":"Viktoras","family":"Masevi\u010dius","sequence":"additional","affiliation":[{"name":"Institute of Biotechnology, Life Sciences Center, Vilnius University , 10257 Vilnius ,","place":["Lithuania"]},{"name":"Institute of Chemistry, Faculty of Chemistry and Geosciences, Vilnius University , 03225 Vilnius ,","place":["Lithuania"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2427-3997","authenticated-orcid":false,"given":"Giedrius","family":"Vilkaitis","sequence":"additional","affiliation":[{"name":"Institute of Biotechnology, Life Sciences Center, Vilnius University , 10257 Vilnius ,","place":["Lithuania"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1395-2030","authenticated-orcid":false,"given":"Saulius","family":"Klima\u0161auskas","sequence":"additional","affiliation":[{"name":"Institute of Biotechnology, Life Sciences Center, Vilnius University , 10257 Vilnius ,","place":["Lithuania"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2026,9,22]]},"reference":[{"key":"2026092204584363500_B1","doi-asserted-by":"publisher","first-page":"204","DOI":"10.1038\/nrg3354","article-title":"DNA methylation: roles in mammalian development","volume":"14","author":"Smith","year":"2013","journal-title":"Nat Rev Genet"},{"key":"2026092204584363500_B2","doi-asserted-by":"publisher","first-page":"339","DOI":"10.1038\/nature10960","article-title":"A unique regulatory phase of DNA methylation in the early mammalian embryo","volume":"484","author":"Smith","year":"2012","journal-title":"Nature"},{"key":"2026092204584363500_B3","doi-asserted-by":"publisher","first-page":"2395","DOI":"10.1093\/hmg\/9.16.2395","article-title":"The DNA methyltransferases of mammals","volume":"9","author":"Bestor","year":"2000","journal-title":"Hum Mol Genet"},{"key":"2026092204584363500_B4","doi-asserted-by":"publisher","first-page":"45","DOI":"10.1007\/978-3-031-11454-0","article-title":"Domain Structure of the Dnmt1, Dnmt3a, and Dnmt3b DNA Methyltransferases","volume-title":"DNA Methyltransferases - 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