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It functions as the initiating factor in uracil base excision repair (UBER) by catalyzing the removal of uracil lesions in genomic DNA, a necessary first step in restoring genome integrity after hydrolytic deamination of cytosine to uracil or misincorporation of deoxyuridine monophosphate during replication. Although methods have been developed to study UBER in vitro and in cellulo, none provide a quantitative readout of UNG activity on the chromosomal DNA of living cells. To address this gap, we created an UNG biosensor (U-report) that utilizes a modified cytosine base editor to generate a targeted genomic uracil lesion in a fluorescent reporter for C-to-U editing activity. UNG activity ablation through uracil DNA glycosylase inhibition (Ugi) or UNG-knockout results in elevated reporter fluorescence. Isoform-specific knockouts show that mitochondrial UNG1 also contributes to UBER of nuclear DNA. Our studies establish a real-time biosensor for quantification of chromosomal DNA uracil excision activity in living cells and, in support of prior studies, indicate that both UNG isoforms should be addressed in small molecule inhibitor development programs.<\/jats:p>","DOI":"10.1093\/nar\/gkag852","type":"journal-article","created":{"date-parts":[[2026,8,19]],"date-time":"2026-08-19T11:53:05Z","timestamp":1787140385000},"source":"Crossref","is-referenced-by-count":0,"title":["Genetic studies with a uracil DNA glycosylase biosensor support a role for mitochondrial UNG1 in nuclear uracil repair"],"prefix":"10.1093","volume":"54","author":[{"given":"Yu-Hsiu T","family":"Lin","sequence":"first","affiliation":[{"name":"Department of Biochemistry and Structural Biology, University of Texas Health San Antonio , San Antonio, TX 78229 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Analisa 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