Two distinct Okazaki fragment failure modes dominate mutagenesis in the absence of flap endonuclease 1

The yeast RAD27 gene encodes the Rad27/Fen1 nuclease, which contributes to genome stability during Okazaki fragment maturation (OFM), DNA mismatch repair (MMR), base excision repair, and other processes. Here, we analyze whole genome mutation accumulation in Saccharomyces cerevisiae lacking RAD27 and show that its loss elevates diverse mutation classes arising through multiple mechanistic pathways. Overall mutation rates in rad27Δ cells are over 60-fold higher than in wild type and only modestly lower than in MMR-deficient strains. However, most mutations in rad27Δ cells cannot be explained by defective MMR, elevated translesion synthesis, or defects in other Rad27-associated repair processes. Instead, mutation spectra implicate aberrant processing of Okazaki fragment intermediates via DNA terminus slippage before ligation (SBL) and template switching (TS). The latter may arise through replication-associated processes such as transient primer relocation (TPR) or through recombination-mediated mechanisms including homoeologous recombination. SBL accounts for the majority of insertion mutations in rad27Δ cells, while TS explains substantial fractions of substitutions, insertions, deletions, and copy number variants, often involving non-local templates. Our results indicate that Rad27 suppresses genome instability through multiple mechanistically distinct roles. Together, these findings reveal Rad27/Fen1 to be a determinant of replication-associated genome stability on par with MMR.