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This stress can drive genomic instability, a cancer hallmark. RNA:DNA hybrids, such as R-loops and single genome-embedded ribonucleotides, are significant sources of replication stress. RNA:DNA hybrid homeostasis must therefore be tightly regulated through prevention and removal. Ribonuclease H2 (RNase H2) functions both in R-loop removal and excision of single ribonucleotides from genomic DNA. Recent research has generated new mechanistic insights into the functions of RNase H2 in the replication stress response, and implicated both loss and overexpression of RNase H2 in cancer development and therapy response. These findings help generate new models but also raise new questions. This Review explores the contribution of RNA:DNA hybrids to replication stress, the involvement of RNase H2 in regulating these structures, and the emerging roles of RNase H2 in replication stress response and cancer.<\/jats:p>","DOI":"10.1242\/jcs.264148","type":"journal-article","created":{"date-parts":[[2025,12,10]],"date-time":"2025-12-10T10:13:09Z","timestamp":1765361589000},"source":"Crossref","is-referenced-by-count":5,"title":["Emerging roles of RNA:DNA hybrid regulation by mammalian ribonuclease H2 in replication stress and cancer"],"prefix":"10.1242","volume":"138","author":[{"given":"Rosanna J.","family":"Wilkins","sequence":"first","affiliation":[{"name":"School of Medical Sciences, College of Medicine and Health, University of Birmingham 1 Department of Cancer and Genomic Sciences , , Birmingham B15 2TT ,","place":["UK"]},{"name":", University of Birmingham 2 Birmingham Centre for Genome Biology , Birmingham B15 2TT ,","place":["UK"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5746-213X","authenticated-orcid":false,"given":"Eva","family":"Petermann","sequence":"additional","affiliation":[{"name":"School of Medical Sciences, College of Medicine and Health, University of Birmingham 1 Department of Cancer and Genomic Sciences , , Birmingham B15 2TT ,","place":["UK"]},{"name":", University of Birmingham 2 Birmingham Centre for Genome Biology , Birmingham B15 2TT ,","place":["UK"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"237","published-online":{"date-parts":[[2025,12,10]]},"reference":[{"key":"2025121005130016800_JCS264148C1","doi-asserted-by":"publisher","first-page":"145","DOI":"10.1053\/j.gastro.2018.09.047","article-title":"Epithelial RNase H2 maintains genome integrity and prevents intestinal tumorigenesis in mice","volume":"156","author":"Aden","year":"2019","journal-title":"Gastroenterology"},{"key":"2025121005130016800_JCS264148C2","doi-asserted-by":"publisher","first-page":"115","DOI":"10.1016\/j.molcel.2012.04.009","article-title":"R loops: from transcription byproducts to threats to genome stability","volume":"46","author":"Aguilera","year":"2012","journal-title":"Mol. 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