The roles of NHEJ and TLS pathways in genomic alterations and phenotypic evolution in the yeast Yarrowia lipolytica

Non-homologous end joining (NHEJ) is a DNA repair pathway that directly ligates broken DNA ends without the need for a homologous template, whereas translesion synthesis (TLS) is a DNA damage tolerance mechanism in which specialized DNA polymerases bypass lesions on the template strand. Although both pathways play critical roles in maintaining genome integrity across organisms, they inherently introduce mutations. Here, we investigate how these two pathways contribute to spontaneous and genotoxic stress-induced genomic alterations in the yeast Yarrowia lipolytica. A NHEJ-deficient mutant (ku70) and three TLS-deficient mutants (rev1, rev3, and rad30) are subjected to mutation accumulation experiments, followed by whole-genome sequencing. Our results show that the deletion of KU70 has no significant effect on the rates of spontaneous single-nucleotide variations (SNVs), small insertions and deletions, or chromosomal rearrangements, while the deletion of REV1 and REV3 leads to significant reductions in spontaneous SNV rates. These findings indicate that TLS but not the NHEJ pathway is a major contributor to spontaneous mutagenesis in Y. lipolytica. Moreover, exposure to 0.02% methyl methanesulfonate and 80 J/m² ultraviolet (UV) radiation resulted in 48- and 107-fold increases in SNV rates, respectively. These induced SNVs were largely dependent on DNA polymerases Rev1 and ζ, further underscoring their central roles in genotoxic stress-induced mutagenesis. We observe that DNA polymerase η can suppress C to T and C to A substitutions while promoting T to C mutations, exhibiting a dual function in regulating mutagenesis under UV treatment. Phenotypic evolution experiments reveal that TLS activity enhances the adaptive potential of Y. lipolytica under oxidative stress, underlying its broader impact on environmental fitness. Together, these findings provide new insights into the distinct roles of the NHEJ and TLS pathways in preserving genome integrity in Y. lipolytica. KEY POINTS: • The NHEJ pathway has a limited role in spontaneous genomic alterations in Y. lipolytica. • DNA polymerases Rev1 and ζ contribute to most UV- and MMS-induced mutations. • The dual roles of Pol η in UV-induced mutations were revealed. • NHEJ and TLS pathways are crucial to phenotypic evolution of Y. lipolytica.