Uracil overload triggers global genome instability and dUTPdysgenesis

Uracil accumulation in DNA is a pervasive threat to genomic integrity, yet the global dynamics of how uracil overload drives genome instability remain elusive. Using Saccharomyces cerevisiae strains with attenuated dUTPase (DUT1) expression, we mapped the landscapes of genomic alterations including point mutations, recombination, and aneuploidy. Deletion of the UNG1 suppressed most induced mutations and loss-of-heterozygosity (LOH), identifying base excision repair as the primary driver of genomic alterations, whereas aneuploidy directly depends on the persistence of intact genomic uracil. Mechanistically, beyond localized bypass of static AP sites by translesion synthesis polymerases, the massive influx of uracil lesions induces a repair bottleneck by depleting Ung1 and Apn1. This resource titration, coupled with downstream processing intermediates, primarily drives mutagenesis at heterologous nonthymine positions. Furthermore, persistent static AP sites preferentially stimulate homologous recombination sub-pathway that yield interstitial LOH, whereas Apn1-mediated processing channels recombination toward terminal LOH. Crucially, we define a novel post-zygotic isolation mechanism, dUTPdysgenesis, where parental wild-type Ung1 unleashes an immediate enzymatic shock against a uracil-preloaded genome upon mating, triggering G1-phase chromosomal shattering and mutations. Collectively, this study transitions the paradigm of uracil mutagenesis from localized errors to a global engine of genome instability, bridging nucleotide metabolism, DNA repair kinetics, and speciation barriers.