{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T11:30:09Z","timestamp":1787052609645,"version":"build-2736575974"},"posted":{"date-parts":[[2026,8,15]]},"group-title":"Molecular Biology","reference-count":75,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,8,15]],"date-time":"2026-08-15T00:00:00Z","timestamp":1786752000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"award":["R35GM134918"],"award-info":[{"award-number":["R35GM134918"]}],"id":[{"id":"https:\/\/ror.org\/04q48ey07","id-type":"ROR","asserted-by":"publisher"}]},{"award":["R35GM133437"],"award-info":[{"award-number":["R35GM133437"]}],"id":[{"id":"https:\/\/ror.org\/04q48ey07","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,8,14]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  DNA helicases preserve genome stability by unwinding DNA during replication, repair, recombination, and transcription, yet their sites of action\n                  <jats:italic>in vivo<\/jats:italic>\n                  remain difficult to define. Here, we describe a sequencing-based strategy to map helicase activity genome-wide by coupling helicases to the single-stranded DNA-specific activation-induced cytidine deaminase (AID). Deamination of cytosines exposed during helicase-mediated DNA unwinding generates strand-specific mutational footprints that can be detected by whole-genome sequencing at near-nucleotide resolution. Using the\n                  <jats:italic>Saccharomyces cerevisiae<\/jats:italic>\n                  RecQ4-family helicase Hrq1, a functional homolog of human RECQL4, we generated the first genome-wide map of Hrq1 activity. Hrq1-dependent deaminations were highly enriched at RNA polymerase III (RNAPIII)-transcribed genes, particularly tRNA genes, where they occurred predominantly on the transcriptional template strand. This localization was reproducible using both overexpressed Hrq1-AID fusions and an inducible dimerization system that recruited AID to endogenously expressed Hrq1, and it was markedly reduced by helicase-inactivating mutation, indicating that active DNA unwinding underlies the observed signal. Hrq1 associated with nearly all tRNA genes irrespective of transcription level, replication orientation, or proximity to transposable elements, yet deletion or overexpression of Hrq1 did not detectably alter pre-tRNA abundance or RNA polymerase III recycling under the conditions tested. Application of the same approach to the PIF1-family helicase Rrm3 recovered its established enrichment at a subset of highly transcribed, head-on tRNA genes, validating the method. Together, these findings establish AID-mediated mutational footprinting as a general approach for mapping helicase activity\n                  <jats:italic>in vivo<\/jats:italic>\n                  and reveal an unexpected, widespread association of the RecQ4-family helicase Hrq1 with RNAPIII-transcribed genes.\n                <\/jats:p>","DOI":"10.64898\/2026.08.13.744683","type":"posted-content","created":{"date-parts":[[2026,8,15]],"date-time":"2026-08-15T08:55:14Z","timestamp":1786784114000},"source":"Crossref","is-referenced-by-count":0,"title":["Genome-wide mapping of helicase-generated ssDNA reveals Hrq1 activity at RNA polymerase III-transcribed genes"],"prefix":"10.64898","author":[{"given":"Shaili","family":"Regmi","sequence":"first","affiliation":[{"name":"Center for Genomics and Systems Biology, Department of Biology, New York University, New York, NY, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Noof","family":"Alsulaiti","sequence":"additional","affiliation":[{"name":"Molecular & Cellular Biochemistry Department, Indiana University, Bloomington, IN, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daniel","family":"Darling","sequence":"additional","affiliation":[{"name":"Center for Genomics and Systems Biology, Department of Biology, New York University, New York, NY, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anastasiya","family":"Bolgova","sequence":"additional","affiliation":[{"name":"Center for Genomics and Systems Biology, Department of Biology, New York University, New York, NY, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bertrand","family":"Theulot","sequence":"additional","affiliation":[{"name":"Center for Genomics and Systems Biology, Department of Biology, New York University, New York, NY, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Spencer J.","family":"Gray","sequence":"additional","affiliation":[{"name":"Molecular & Cellular Biochemistry Department, Indiana University, Bloomington, IN, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Matthew L.","family":"Bochman","sequence":"additional","affiliation":[{"name":"Molecular & Cellular Biochemistry Department, Indiana University, Bloomington, IN, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0898-8629","authenticated-orcid":false,"given":"Duncan J.","family":"Smith","sequence":"additional","affiliation":[{"name":"Center for Genomics and Systems Biology, Department of Biology, New York University, New York, NY, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026081804200477000_2026.08.13.744683v1.1","doi-asserted-by":"crossref","first-page":"e963429","DOI":"10.4161\/23723548.2014.963429","article-title":"Roles of DNA helicases in the maintenance of genome integrity","volume":"1","year":"2014","journal-title":"Mol Cell Oncol"},{"key":"2026081804200477000_2026.08.13.744683v1.2","first-page":"1","article-title":"Helicases: an overview","volume":"587","year":"2010","journal-title":"Methods Mol Biol"},{"key":"2026081804200477000_2026.08.13.744683v1.3","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2008.08.027"},{"key":"2026081804200477000_2026.08.13.744683v1.4","doi-asserted-by":"publisher","DOI":"10.1091\/mbc.E11-01-0045"},{"key":"2026081804200477000_2026.08.13.744683v1.5","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-genet-102209-163602"},{"key":"2026081804200477000_2026.08.13.744683v1.6","doi-asserted-by":"publisher","DOI":"10.1038\/nrc2682"},{"key":"2026081804200477000_2026.08.13.744683v1.7","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-biochem-060713-035428"},{"key":"2026081804200477000_2026.08.13.744683v1.8","doi-asserted-by":"publisher","DOI":"10.1016\/j.canlet.2017.10.021"},{"key":"2026081804200477000_2026.08.13.744683v1.9","doi-asserted-by":"publisher","DOI":"10.1186\/1750-1172-5-2"},{"key":"2026081804200477000_2026.08.13.744683v1.10","unstructured":"Van Maldergem, L. , Piard, J. , Larizza, L. and Wang, L.L. 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