Mitochondrial uracil DNA glycosylase contributes to nuclear base excision repair

The universally conserved enzyme uracil-DNA N-glycosylase (UNG) plays a central role in maintaining genome stability. It serves 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 deoxy-uridine 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 ablation through uracil DNA glycosylase inhibitor (Ugi) or UNG-knockout results in elevated reporter florescence. Surprisingly, isoform-specific knockouts reveal that mitochondrial UNG1 also contributes to UBER of nuclear DNA. Our studies combine to establish a real-time biosensor for quantification of chromosomal DNA uracil excision activity in living cells and indicate that both UNG isoforms should be considered in small molecule inhibitor development programs.