This study systematically investigated the genomic alterations in Saccharomyces cerevisiae driven by Replication Factor A (RFA) dosage insufficiency using a promoter-replacement strategy combined with mutation accumulation and whole-genome sequencing. Our findings reveal that transcriptional suppression of RFA2 or RFA3 leads to severe growth inhibition. RFA deficiency induces a distinct mutational spectrum characterized by a high frequency of monosomy and terminal deletions, indicative of severe replication stress. Furthermore, loss of heterozygosity is significantly enriched at centromeres and high-GC regions, underscoring the role of RFA in stabilizing intrinsic genomic barriers. Utilizing an APOBEC3B-induced mutagenesis assay, we demonstrate that RFA insufficiency leads to the extensive accumulation of exposed ssDNA with a distinct bias towards the lagging strand template. Notably, we observed that cells spontaneously inactivate Mismatch Repair (MMR) genes, such as MSH2 and PMS1, to survive RFA-induced stress. This hypermutant phenotype grants a certain degree of growth recovery on Low Galactose (LG) medium. Overall, these findings demonstrate that RFA dosage is a key determinant of genomic integrity and elucidate how repair pathway modulation drives adaptive evolution under replication stress.

Superoxide dismutase 1 (Sod1) is long recognized as a frontline antioxidant, yet its broader role in safeguarding the eukaryotic genome remains under-explored. Here, using a yeast model, we reveal that Sod1 is an indispensable guardian of genomic integrity. Its absence unleashes a genomic storm: whole-genome sequencing uncovered a dramatic surge in single nucleotide variations (13-fold), loss of heterozygosity (12-fold), chromosomal rearrangements (4-fold), and a staggering 48-fold increase in aneuploidy. Remarkably, this genomic instability was completely abolished under anaerobic conditions, identifying reactive oxygen species as the sole executioner. We demonstrate that Sod1 deficiency sculpts a unique mutational landscape dominated by C:G to A:T transversions—a molecular fingerprint of 8-oxoguanine overproduction. Mechanistically, these lesions are exacerbated by error-prone DNA polymerase ζ but mitigated by the concerted actions of Ogg1-mediated excision and DNA polymerase η. Critically, we show that Amyotrophic Lateral Sclerosis-associated variants are not created equal: the H48Q mutation shatters genome stability, whereas others (A4V, G36R, and G93A) remain benign in this context. Unlike yeast Sod1, human Sod1 is highly resistant to amino acid substitutions outside copper-binding sites, thus preventing the loss of enzyme function. Overall, our findings provide a mechanistically framework for understanding how Sod1 dysfunction drives genome instability in eukaryotic cells.