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

Abstract

Abstract

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.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00253-025-13575-2.

Introduction

Non-homologous end joining (NHEJ) is a critical DNA repair pathway responsible for repairing DNA double-strand breaks (DSBs) in eukaryotes. Unlike homologous recombination (HR), which requires a homologous sequence as a template for repair, NHEJ directly ligates the broken DNA ends without the need for sequence homology (Daley et al. 2005). In the NHEJ process, the Ku70/Ku80 heterodimer recognizes and binds to the broken DNA ends, forming a protective cap to prevent further degradation (Fell and Schild-Poulter 2015). Ku recruits the DNA-dependent protein kinase catalytic subunit, which facilitates the bridging of DNA ends. While efficient in repairing a wide range of DSBs, NHEJ often introduces small insertions or deletions (InDels) at the repair site (Lemos et al. 2018). NHEJ has been harnessed in synthetic biology for precise genome editing using CRISPR-Cas systems (Fraczek et al. 2018). By inducing DSBs at specific loci, previous studies have exploited NHEJ to create targeted InDels for functional studies and biotechnological applications (Cui et al. 2021; Ploessl et al. 2022).

Translesion synthesis (TLS) is a DNA damage tolerance mechanism that allows cells to bypass replication-blocking DNA lesions, a conserved pathway from the single-cell organism yeast to humans (Prakash et al. 2005). While this mechanism prevents replication fork collapse and ensures genome duplication, it often introduces DNA mutations (Kiktev et al. 2018). In the model yeast Saccharomyces cerevisiae, TLS DNA polymerases include Rev1, polymerase η (Pol η), and polymerase ζ (Pol ζ) (Johnson et al. 2006). Rev1 is a specialized TLS polymerase that incorporates a cytosine opposite abasic sites or certain damaged bases (Auerbach and Demple 2010). Apart from its catalytic role, Rev1 acts as a scaffold protein, recruiting other TLS polymerases like Pol ζ to the site of DNA damage. Pol ζ, composed of the Rev3 catalytic subunit and Rev7 regulatory subunit, extends the DNA strand beyond the lesion after other TLS polymerases insert a nucleotide opposite the damage (Badugu et al. 2024; Washif et al. 2024). Pol η efficiently bypasses UV-induced DNA lesions and minimizes mutations compared to other TLS polymerases (Kozmin et al. 2003; Vandenberg et al. 2023).

Yarrowia lipolytica is a non-conventional yeast that has been isolated from diverse environments, including dairy products, petroleum derivatives, and marine ecosystems (Park and Ledesma-Amaro 2023). Its remarkable ability to metabolize a wide range of substrates—particularly hydrophobic compounds—and to produce lipids and value-added chemicals at high yields makes it a promising host for various industrial biotechnology applications (Liu et al. 2022, 2024; Ma et al. 2022; Cui et al. 2023). Despite these advances, our understanding of the genomic evolution of Y. lipolytica and the development of its genetic tools remains limited compared to the extensively studied yeast S. cerevisiae. In the genetic engineering of Y. lipolytica, the inactivation of the NHEJ pathway—typically through deletion of KU70—has been widely employed to reduce the risk of random DNA integration (Shen et al. 2024; Liu et al. 2025). However, the impact of NHEJ deficiency on genome integrity has not been thoroughly characterized. Likewise, although sequence homology analyses have confirmed the presence of key translesion synthesis (TLS) DNA polymerases—Rev1, Pol η, and Pol ζ—in Y. lipolytica, their genetic roles and contributions to genome evolution and mutagenesis remain poorly understood.

In this study, we conducted a mutation accumulation (MA) experiment on wild-type Y. lipolytica strain as well as NHEJ- and TLS-deficient mutants under both spontaneous and mutagenic conditions. Whole-genome sequencing of the MA lines revealed the distinct roles of these two pathways in shaping DNA mutation rates and mutational spectra in Y. lipolytica. Furthermore, a phenotypic evolution experiment demonstrated that TLS is essential for enhancing stress tolerance over time. Our findings enhance our understanding of the roles of NHEJ and TLS pathways in genome evolution in Y. lipolytica.

Materials and methods

Yeast strains and medium

The Y. lipolytica strains used in this study, along with their genotypes, are detailed in Table 1. The strains were normally cultured in YPD medium, consisting of 10 g/L yeast extract, 20 g/L peptone, and 20 g/L dextrose. Twenty grams per liter of agar was added to make solid medium.

Table 1.

Y. lipolytica strains used in this study

Strains	Genotype	Source
PO1f	MatA leu2-270 ura3-302 xpr2-322 axp1-2	Madzak et al. (2000)
PPF	MatA leu2-270 ura3-302 xpr2-322 axp1-2 int::CAS9-LEU2 LSY9	Xiong et al. (2025)
PPY3	MatA leu2-270 ura3-302 xpr2-32 axp1-2 int::CAS9-LEU2 LYS9-Δ	Xiong et al. (2025)
ku70	MatA leu2-270 ura3-302 xpr2-322 axp1-2 int::CAS9-LEU2 LYS9-Δ KU70Δ∷LYS9	Xiong et al. (2025)
rev1	MatA leu2-270 ura3-302 xpr2-322 axp1-2 int::CAS9-LEU2 LYS9-Δ REV1Δ∷LYS9	Xiong et al. (2025)
rev3	MatA leu2-270 ura3-302 xpr2-322 axp1-2 int::CAS9-LEU2 LYS9-Δ REV3Δ∷LYS9	Xiong et al. (2025)
rad30	MatA leu2-270 ura3-302 xpr2-322 axp1-2 int::CAS9-LEU2 LYS9-Δ RAD30Δ∷LYS9	Xiong et al. (2025)

MA experiment of Y. lipolytica strains

To investigate spontaneous genomic alterations in Y. lipolytica, we subcultured nine independent MA lines of Y. lipolytica strains on solid YPD medium for 85 generations. During each cycle of subculture, a colony on the YPD plate was randomly selected and stroked on a new YPD plate, followed by incubation at 30 °C for 2 days for each generation. For UV treatment, 10 MA lines of each strain were exposed to 254 nm UV light at an intensity of 80 J/m² after streaking for 90 s, followed by incubation at 30 °C for 2 days. For MMS treatment, 10 MA lines of Y. lipolytica strains were stroked on YPD plates containing 0.02% MMS and then incubated at 30 °C for 3 days. The MA lines treated with UV and MMS treatments were subcultured for ten generations.

DNA extraction and genome sequencing

Yeast cells were cultured overnight in 5 mL of YPD medium to prepare genomic DNA, which was extracted using the Genomic DNA Extraction Kit (Omega Bio-tek Inc., GA, USA). The quality and concentration of the extracted DNA were assessed using the Qubit™ dsDNA BR Assay Kit (Thermo Fisher Scientific, MA, USA) and agarose gel electrophoresis. For library construction, index adapters from the MGI Adapter Set 8 (Vazyme, Nanjing, China) were ligated to the genomic DNA. Library preparation was carried out on the MGISP-960 automated platform (MGI, Shenzhen, China) using the VAHTS® Universal Plus DNA Library Prep Kit for MGI (Vazyme, Nanjing, China), and the resulting libraries were quantified and assessed for quality using the Qubit™ dsDNA HS Assay Kit (Thermo Fisher Scientific, MA, USA). Circularization of the libraries was performed with the VAHTS® Circularization Kit for MGI (Vazyme, Nanjing, China), followed by the generation of DNA nanoballs (DNBs) using the MGISEQ-2000RS High Throughput Sequencing Kit (MGI, Shenzhen, China). DNBs were loaded onto the MGISEQ-2000 sequencing platform (MGI, Shenzhen, China) and sequenced using a paired-end 2 × 150 bp strategy. Raw sequencing reads were subjected to quality control using FastQC (v0.11.9) and trimmed with Trimmomatic (v0.39) to remove adapter sequences and bases with a Phred quality score below 20.

Detection of single-nucleotide variations (SNVs) and InDels

High-quality reads (~ 2G for each sample) were aligned to the Y. lipolytica reference genome (GCF_001761485.1; https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_001761485.1/) using the BWA-MEM algorithm with default parameters (Li and Durbin 2009). The resulting SAM files were converted to BAM format and sorted using SAMtools (Li et. al. 2009). SNVs and InDels were identified using VarScan (Koboldt et al. 2012). VarScan performs comparably to other variant callers such as FreeBayes and GATK for mutation detection in yeast, but has been more frequently used in our previous studies (Zheng et al. 2016; Zhang et al. 2022; Xiong et al. 2025). Mutations detected in all isolates of the wild-type strain and each mutant were considered pre-existing and excluded from further analysis. Furthermore, mutations supported by fewer than 30 reads or by less than 80% of the reads covering the site were filtered out; for duplicated regions, a lower threshold of 40% was applied. Variant annotation was performed with SnpEff (Cingolani et al. 2012), utilizing a pre-annotated Y. lipolytica genome database to predict the potential effects of each variant on coding sequences and regulatory regions.

Mutation rates ${\mu }_{\mathit{bp}}$ per base pair per cell division were calculated as follows: ${\mu }_{\mathit{bp}}=\frac{n_{\mathit{bp}}}{N\times {\mathit{gen}}_{\mathit{tot}}\times t}$, where nbp is the number of mutations of any type of SNVs or InDels, N is 20,500,000 (the number of base pairs of the genome of Y. lipolytica), gen_tot is the total number of subculture generations, and t is the number of divisions from a cell to form a colony (Xiong et al. 2025).

Analysis of DNA copy number variations

The sequencing coverage at each base position across the genomes of Y. lipolytica isolates was calculated using bedtools (Quinlan and Hall 2010), with sorted BAM files as input. To assess coverage patterns, the genome was partitioned into bins of 2000 bp with a sliding window of 1000 bp, and the depth of coverage for each bin was computed using a custom Python script (see Supplemental Text). The resulting coverage data were then used for graphical visualization using a custom R script, following the approach described by Bai et al. (2025).

Minimum inhibitory concentration (MIC) determination

To determine the MIC₅₀ for all strains and stressors (Zeocin and H₂O₂) use, a single colony was inoculated and grown to an OD of 0.5 to 1 and diluted to an OD of 0.01 in culture media containing the indicated concentration of a stressor. Cells were grown at 30 °C for 16 h, and the OD of each culture was measured.

Phenotypic evolution assay

A single colony of the Y. lipolytica strains was grown in liquid YPD (5 mL) until an OD of ~ 2 was reached. Culture was then diluted to an OD of 0.01 in liquid YPD and grown in seven different concentrations of the indicated stressors, ranging from no stressor to 16 × the MIC. Yeast cells were grown for 24 h at 30 °C with aeration, after which the OD was measured. The culture exposed to the highest concentration of the stressor that still exhibited an OD greater than half that of the control was diluted 100-fold to an approximate OD of 0.01 and then regrown in seven different stressor concentrations. This subculture process was repeated three times.

Statistical analysis

The 95% confidence intervals for mutation rates were estimated based on a Poisson distribution. To assess the statistical significance of differences in mutation rates between spontaneous and mutagen-induced conditions, the Wilcoxon rank-sum test was applied. A P value less than 0.05 was considered statistically significant.

Results

The contribution of NHEJ and TLS pathways to spontaneous genomic alterations in Y. lipolytica

To investigate the spontaneous genomic alterations of Y. lipolytica, nine wild-type isolates derived from strain PO1f (Table 1) were subcultured on solid YPD medium for 85 generations to accumulate mutations (Fig. 1A). Whole-genome sequencing of these nine isolates identified 62 SNVs (Dataset S1-1) and 37 InDels (Dataset S1-2). Using the formula described in the “Materials and methods” section, we calculated that spontaneous rates of SNVs and InDels were 1.8 × 10⁻¹⁰ (62/9/25/85/20,500,000) and 9.4 × 10⁻¹¹ (37/9/25/85/20,500,000) per base per cell division, respectively (Fig. 1B). These values are comparable to those previously reported for the wild-type Y. lipolytica strain with a W29 genetic background (Xiong et al. 2025), suggesting minimal strain-specific variation in spontaneous mutation rates. C:G to T:A (C to T or G to A) and C:G to A:T were the prominent types of base substitution, accounting for up to 42% and 25% of all SNVs, respectively (Fig. 1C). C:G to T:A substitutions commonly arise from the spontaneous deamination of 5-methylcytosine (5mC) to thymine, which generates a G:T mismatch (Chatterjee and Walker 2017). If this mismatch escapes repair prior to DNA replication, it can result in a fixed C:G to T:A transition. In contrast, C:G to A:T mutations are often associated with oxidative damage to guanine, particularly the formation of 8-oxoguanine (Lynch et al. 2008). This lesion can mispair with adenine during DNA replication, leading to a G:C to T:A transversion, manifested as a C:G to A:T substitution.

Fig. 1.

Mutation accumulation experiment in wild-type strains and mutants defective in non-homologous end joining (NHEJ) and translesion synthesis (TLS) pathways. A The wild type (WT) and mutants ku70, rev1, rev3, and rad30 were subcultured on YPD plates under UV- and MMS-treated conditions for multiple cycles. Nine or ten isolates for each strain were sequenced for mutation detection. B The spontaneous rates of SNVs and InDels of the wild type and mutants ku70, rev1, rev3, and rad30. The asterisk (*) indicates a significant difference from the WT at the 0.05 level (Mann–Whitney test). C Mutation spectrum (transitions and transversions) in the wild type and mutants ku70, rev1, rev3, and rad30

Among the detected InDels, 1-bp deletions were the most prevalent class, accounting for 56.8%, followed by 1-bp insertions at 28.6% (Dataset S1-2). Sequence context analysis revealed that over 90% of these InDels occurred within mononucleotide repeats or microsatellite regions, suggesting that replication slippage, rather than the NHEJ pathway, is the primary mechanism underlying these mutations. To test this hypothesis, we performed whole-genome sequencing on nine isolates derived from a NHEJ-deficient (ku70) mutant that had undergone 85 generations of subculturing. Our previous study demonstrated that deletion of KU70 in Y. lipolytica effectively inactivates the NHEJ pathway and prevents the majority of InDels induced by DNA-damaging agent Zeocin (Xiong et al. 2025). Here, the rate of InDels in these ku70 isolates subcultured under spontaneous conditions was estimated at 7.1 × 10⁻¹¹ (28/9/25/85/20,500,000) per base per cell division, which was not significantly different from that observed in wild-type isolates (Mann–Whitney test, P > 0.05; Fig. 1B). These results confirm that the NHEJ pathway plays a minimal role in the generation of spontaneous small-scale InDels in Y. lipolytica.

To assess the role of the TLS pathway in spontaneous mutagenesis, we sequenced ten subcultured isolates from each Y. lipolytica mutant deficient in TLS polymerases: rev1, rev3, and rad30 (Xiong et al. 2025). We found that the deletion of REV1 (encoded by YALI1_F12757g, spanning base pairs 1,275,748–1,279,407 on chromosome F) and REV3 (encoded by YALI1_C29926g, spanning base pairs 2,992,626–2,996,642 on chromosome C) resulted in 50% and 40% reductions in SNV rates, respectively (Mann–Whitney test, P < 0.05; Fig. 1B). These results indicate that half of spontaneous SNVs are attributable to the activity of the TLS pathway involving Rev1 and DNA Pol ζ. In contrast, deletion of RAD30 (encoded by YALI1_C02152g, base pairs 213,307–215,229 on chromosome C), which encodes DNA Pol η, did not significantly affect the overall SNV rate. Furthermore, the relative proportions of specific base substitutions—including two transitions (C:G to T:A and T:A to C:G) and four transversions (C:G to T:A, A:T to C:G, C:G to G:C, and A:T to T:A)—remained unchanged in the absence of NHEJ or TLS polymerase activity under spontaneous conditions (Fisher’s exact test, P > 0.05, Fig. 1C).

In addition to point mutations, we identified three large duplications among the nine sequenced wild-type isolates: from 1630 to 2108 kb on chromosome E (Fig. 2A), 413 to 2251 kb on chromosome A (Fig. 2B), and 1209 to 1257 kb on chromosome C (Fig. 2C). The frequency of such large-scale rearrangements was estimated at 1.6 × 10⁻⁴ events per cell division. Similarly, three DNA fragment duplications were detected in the nine ku70 mutant isolates (Fig. 2D–F), indicating that the deficiency of the NHEJ pathway does not significantly impact the spontaneous rate of chromosomal rearrangement in Y. lipolytica. In the rad30, rev1, and rev3 mutants, we detected 2, 1, and 1 large duplications, respectively. However, the small number of observed events prevents us from drawing definitive conclusions regarding the potential suppression of large-scale rearrangements in these TLS-deficient mutants.

Fig. 2.

Chromosomal rearrangements in the wild-type isolates and ku70mutant. Through calculating the sequencing coverage, three DNA duplications were identified in the isolates of wild type (A–C) and ku70 mutant (D–F), respectively. Yyl-1 and Yyl-3 are isolates of the wild-type Y. lipolytica strain, whereas YK3, YK9, and YK10 are isolates derived from the ku70 mutant background

MMS-induced DNA mutations in wild-type strains and NHEJ- and TLS-deficient mutants

MMS is a well-known alkylating agent that introduces alkyl groups to DNA bases, particularly at the O6 and N7 positions of guanine (O⁶-MeG and N⁷-MeG) and the N3 position of adenine (N³-MeA), leading to the formation of unstable DNA adducts (Volkova et al. 2020). MMS has been widely used to study DNA repair mechanisms and for strain improvement in industrial applications (Dong et al. 2022). Under 0.02% MMS treatment, the ku70 and rad30 mutants exhibited biomass formation comparable to that of the wild-type strain after 24 h of incubation, while the rev1 and rev3 mutants displayed significantly slower growth (Fig. S1A in Supplemental file). To assess the mutagenesis of MMS in Y. lipolytica, we subcultured ten isolates of the wild-type strain PPF and NHEJ and TLS mutants on solid YPD containing 0.02% MMS for ten generations, followed by whole-genome sequencing. We identified 431 SNVs and 14 InDels among the ten isolates of wild-type strains treated with MMS, indicating their rates being 8.4 × 10⁻⁹ and 2.7 × 10⁻¹⁰ per base per cell division (Fig. 3A). These rates were 47- and threefold higher than that in the untreated isolates.

Fig. 3.

MMS-induced mutations in wild-type isolates and mutants defective in NHEJ and TLS pathways. A The rates of SNVs and InDels of the wild-type strains and mutants rev1, rev3, and rad30 treated with MMS. The asterisk (*) denotes statistically significant differences (P < 0.05) compared to the WT, based on the Mann–Whitney U test. B Mutation spectrum in the wild-type strains and mutants rev1, rev3, and rad30. C Three-base motifs for spontaneous SNVs (top) and MMS-induced SNVs (bottom)

In ku70-deficient cells, the frequency of MMS-induced SNVs was reduced by approximately 40% relative to wild-type controls (Fig. 3A). A comparable decrease (37%) in SNV rate was observed upon deletion of RAD30, suggesting that both Ku70 and Rad30 contribute to the accumulation of MMS-induced mutations (Fig. 3A). Strikingly, the disruption of either REV1 or REV3 resulted in a dramatic reduction in MMS-induced mutagenesis (Fig. 3A). Specifically, the SNV rates in the rev1 and rev3 mutants were 7.2 × 10⁻¹⁰ and 8.8 × 10⁻¹⁰ per base per cell division, respectively—representing only 8.6% and 10% of the rate observed in MMS-treated wild-type strains (Fig. 3A). These results strongly support the conclusion that Rev1 and DNA Pol ζ constitute the primary pathway responsible for MMS-induced base substitution events.

In addition to increasing the overall mutation burden, MMS treatment induced a marked shift in the mutational spectrum. Specifically, A:T to T:A transversions became the predominant substitution type, comprising up to 56% of all SNVs following exposure (Fig. 3B). Analysis of the bases adjacent to MMS-induced A:T to T:A substitutions suggests a preference for G/C over A/T as the 5′ base of the mutated nucleotide (Fig. 3C; P < 0.05, Fisher’s exact test). This sequence context bias was not observed in spontaneous A:T to T:A substitutions (Fig. 3C). Additionally, the relative ratio of A:T to G:C was also significantly elevated by MMS treatment (Fig. 3B; P < 0.05, Fisher’s exact test). These striking enrichments represent a hallmark of MMS-induced DNA damage (particularly N³-MeA) and reflect its preferential resolution via error-prone repair pathways. Deletion of KU70 or RAD30 had no appreciable effect on the MMS-induced mutational profile. In contrast, loss of REV1 or REV3 effectively abolished this mutation signature, with the relative frequency of A:T to T:A transversions reduced to ~ 20% in both mutants (Figs. 3B and 4A). These findings underscore the essential role of Rev1 and DNA Pol ζ in shaping the mutational landscape associated with MMS-induced DNA lesions.

Fig. 4.

The effect of REV1 and REV3 deletion on base substitutions in MMS-treated strains. A The rates of each individual base substitution in the wild-type strain and rev1 and rev3 mutants. B A model depicting the role of Rev1 and Pol ζ in the mutagenesis of MMS

Based on the above results, we propose a working model to illustrate the mechanism of MMS-induced mutagenesis in Y. lipolytica, as depicted in Fig. 4B. In this model, O⁶-MeG and N³-MeA are considered the primary mutagenic lesions. O⁶-MeG is a highly mutagenic adduct that mispairs with thymine during DNA replication instead of its canonical partner cytosine. If this mispair escapes repair prior to the next round of replication, it results in G:C to A:T transition mutations. Given that deletion of REV1 and REV3 only modestly reduced the frequency of C:G to T:A mutations, it is likely that the insertion of thymine opposite O⁶-MeG is predominantly carried out by the replicative DNA Pol δ and Pol ε, rather than by the TLS pathway. In contrast, N³-MeA is a replication-blocking lesion that interferes with base pairing by disrupting the N3 position of the adenine ring—an essential site for hydrogen bonding in the DNA duplex. This damage stalls the progression of replicative polymerases δ and ε. To bypass this blockage, Y. lipolytica cells recruit the TLS polymerases Rev1 and Pol ζ, which insert nucleotides in an error-prone manner. Our data suggest that adenine is the most frequently inserted nucleotide opposite N³-MeA, consistent with the predominance of A to T transversions observed in MMS-treated cells.

The roles of TLS and NHEJ pathways in UV-induced mutations in Y. lipolytica

UV radiation can directly damage DNA by inducing the formation of photoproducts, such as cyclobutane pyrimidine dimers (CPDs) and 6–4 photoproducts (6-4PPs), which disrupt the normal structure of the DNA helix (de Lima-Bessa et al. 2008). To investigate the mutagenic effects of UV exposure on Y. lipolytica, ten isolates derived from wild-type, NHEJ-deficient, and TLS-deficient strains were subjected to ten cycles of UV treatment at a dose of 80 J/m² (Fig. 1A). At this UV dosage, the viability of wild-type Y. lipolytica strain PPF was approximately 86% (Fig. S1B). In contrast, the ku70, rad30, rev1, and rev3 mutants exhibited markedly reduced viabilities of 49%, 34%, 16%, and 15%, respectively (Fig. S1B). In UV-treated wild-type isolates, we identified a total of 964 SNVs and 18 InDels (Datasets S1 and S2). Compared to spontaneous conditions (Fig. 1B), UV exposure resulted in a 107-fold increase in the SNV rate (1.9 × 10⁻⁸ per base per cell division) and a 3.7-fold increase in the InDels rate (3.5 × 10⁻¹⁰ per base per cell division) (Fig. 5A). These findings suggest that UV radiation predominantly induces base substitutions rather than small InDels in Y. lipolytica, consistent with our recent study conducted in a different genetic background (Xiong et al. 2025). Notably, deletion of REV1 and REV3 significantly attenuated UV-induced mutagenesis, resulting in 85% and 80% reductions in SNV rates, respectively (Mann–Whitney test, P < 0.05; Fig. 5A). These results demonstrate that the majority of UV-induced SNVs are dependent on the TLS polymerases Rev1 and Pol ζ. In contrast, ku70 and rad30 mutants exhibited 1.3- and 1.4-fold higher SNV rates, respectively, compared to the wild-type strains (Fig. 5A), indicating that both Ku70 and Pol η (Rad30) contribute to the suppression of UV-induced mutations.

Fig. 5.

UV-induced mutations in wild-type strains and mutants defective in NHEJ and TLS pathways. A The rates of SNVs and InDels of the wild-type strains and mutants rev1, rev3, and rad30 treated with UV. The asterisk (*) indicates a significant difference from the WT at the 0.05 level, as determined by the Mann–Whitney U test. B Mutation spectrum in the wild-type strains and mutants rev1, rev3, and rad30. C Three-base motifs for UV-induced SNVs

Under spontaneous conditions, A:T to G:C substitutions accounted for only 8.7% of SNVs (Fig. 5B). Upon UV treatment, however, the frequency of this substitution type increased significantly to 24% in the wild-type Y. lipolytica strain (P < 0.05, Fisher’s exact test), representing the most strongly elevated base change. UV exposure also markedly increased the proportion of C:G to T:A substitutions, which rose to 56% (P < 0.05, Fisher’s exact test; Fig. 5B). Notably, this UV-induced increase in C:G to T:A substitutions was further amplified in mutants lacking RAD30, REV1, or REV3, indicating that these TLS polymerases play a key role in modulating the mutational spectrum under UV stress. In contrast, deletion of KU70 had no significant effect on either the SNV spectrum or InDels frequency (Fig. 5A, B).

Pol η avoids C to T and C to A mutations but contributes to T to C in UV-treated Y. lipolytica

Although the deletion of RAD30 moderately increased the overall SNV rate in UV-treated Y. lipolytica cells, its effects on specific base substitution types were distinct. Notably, the rates of C:G to T:A and C:G to A:T substitutions were elevated by 1.7- and 4.4-fold, respectively, in the rad30 mutant compared to that in the wild-type strain (Mann–Whitney test, P > 0.05; Fig. 6A). In contrast, the frequency of A:T to G:C substitutions was reduced by approximately 65% (Mann–Whitney test, P > 0.05; Fig. 6A). These findings underscore the dual role of DNA Pol η in the UV-induced mutagenic response of Y. lipolytica—acting both to suppress and to generate specific mutation types depending on the lesion context.

Fig. 6.

The effect of RAD30 deletion on base substitutions in UV-treated strains. A The rates of each individual base substitution in the wild-type strain and rad30 mutant. The asterisk (*) indicates a significant difference at the 0.05 level compared to WT. B A model depicting the role of Rev1, Pol ζ, and Pol η in the mutagenesis of UV in Y. lipolytica

UV irradiation predominantly induces CPDs, particularly at dipyrimidine sites such as TT, TC, CT, and CC (Premi et al. 2015). C:G to T:A and C:G to A:T substitutions are generally associated with cytosine-containing CPDs (TC or CC), where cytosine deamination or mispairing may occur during error-prone bypass in the absence of Pol η (Washington et al. 2001). Analysis of the adjacent base of SNVs confirmed that most mutations in UV-treated Y. lipolytica cells were associated with CPDs (Fig. 5C). The increased frequency of these mutations in the rad30 mutant suggests that alternative translesion polymerases, such as Rev1 or Pol ζ, may bypass these lesions in a more error-prone manner when Pol η is absent.

In Fig. 5C, we observed that the majority of T to C mutations occurred within 5′-TT-3′ or 5′-CT-3′ sequence contexts. This pattern suggests that these base substitutions may be associated with CPDs or 6-4PPs. Given that previous studies have shown that DNA Pol η in S. cerevisiae can introduce T to C substitutions during replication across site-specific 6-4PPs (Bresson and Fuchs 2002), we propose that the T to C substitutions observed in UV-treated Y. lipolytica are likewise the result of error-prone bypass of 6-4PPs mediated by Pol η (Fig. 6B). Together, these observations highlight the lesion-type-specific roles of Pol η in Y. lipolytica, balancing accurate translesion synthesis and mutagenesis under UV-induced DNA damage.

Evolved resistance to oxidative stress in Y. lipolytica requires NHEJ and TLS

Our above analysis confirmed the distinct contributions of NHEJ and TLS polymerases to spontaneous and genotoxic stress–induced DNA mutations. We then considered whether decreasing the activity of NHEJ and TLS in cells would affect the kinetics of phenotypic evolution. To test this hypothesis, we utilized a laboratory evolution assay as described in the “Materials and methods” section. During this assay, we measured the adaptation to two oxidative stressors, Zeocin and H₂O₂, in the wild-type and ku70, rev1, rev3, and rad30 mutants. Although all tested strains showed gradually increased MIC₅₀ values during the 7-day evolution, we observed that the wild-type strain PPF and rad30 mutants evolved more rapidly than the ku70, rev1, and rev3 mutants in Zeocin-containing medium (Fig. 7A). Zeocin, a member of the bleomycin antibiotic family, binds directly to DNA and induces both strand breaks and oxidative base damage (Chen and Stubbe 2005; Zheng et al. 2022). Our previous study demonstrated that Ku70 and DNA Pol ζ are responsible for approximately 80% of Zeocin-induced InDels and 67% of SNVs in Y. lipolytica (Xiong et al. 2025). These findings suggest that the reduced mutagenic capacity of NHEJ- and TLS-deficient mutants may impair their ability to generate adaptive genetic variations under Zeocin-induced stress, thereby limiting their evolutionary potential. Similarly, under H₂O₂ stress, rev1, rev3, and ku70 mutants exhibited a lower tendency to evolve increased resistance compared to the wild-type strain and rad30 mutant (Fig. 7B). These results demonstrated the contribution of NHEJ and TLS in the phenotypic evolution of Y. lipolytica under oxidative stress.

Fig. 7.

Resistance evolution of Y. lipolytica strains under Zeocin and H₂O₂stress. MIC₅₀ values of the wild-type strain PPF and the mutants rev1, rev3, and rad30 during the continuous passages in the presence of A Zeocin and B H₂O₂

Discussion

In this study, we employed a MA experiment combined with whole-genome sequencing to systematically investigate the contributions of NHEJ and TLS to both spontaneous and genotoxic stress–induced mutagenesis in Y. lipolytica. Our key findings are as follows: (1) DNA polymerases Rev1 and Pol ζ contribute partially to the accumulation of spontaneous SNVs; (2) both spontaneous and MMS- or UV-induced InDels are primarily generated through replication slippage mechanisms rather than via the NHEJ pathway; (3) Rev1 and Pol ζ are responsible for the majority of SNVs induced by MMS and UV exposure; (4) Pol η suppresses UV-induced C to T and C to A substitutions but promotes T to C mutations, indicating its dual role in lesion-specific mutagenesis; and (5) NHEJ and TLS pathways play a critical role in promoting the evolution of stress robustness in Y. lipolytica. In the following sections, we discuss the mechanistic implications of these findings and their relevance to adaptive evolution.

NHEJ pathway plays very limited role in spontaneous as well as UV- and MMS-induced mutations

Deletion of KU70 is commonly utilized in fungal systems as a genetic strategy to enhance the efficiency of targeted homologous recombination by disabling the NHEJ pathway (Goins et al. 2006; Feng et al. 2012; Verbeke et al. 2013). Despite its widespread application in strain engineering, the potential impact of KU70 deletion on overall genome stability has remained largely unexplored. Here, we systematically evaluated the genomic consequences of KU70 deletion in Y. lipolytica through whole-genome sequencing. Our results demonstrate that KU70 deletion does not significantly affect the rate of spontaneous genomic alterations, including SNVs, InDels, and chromosomal rearrangements (Figs. 1 and 2). These findings suggest that the disruption of the NHEJ pathway alone does not inherently compromise genome integrity under normal growth conditions. Interestingly, under conditions of DNA damage stress—specifically following treatment with MMS or UV radiation—the SNV mutation rates in ku70 mutants exhibited modest but variable changes, showing either slight increases or decreases compared to wild-type strains (Figs. 3A and 5A). Notably, KU70 deletion did not lead to alterations in the SNV mutational spectrum or in the frequency of InDels, indicating that the NHEJ pathway does not directly contribute to the mutagenic processing of these lesions. We hypothesize that the observed fluctuations in mutation rates under UV- and MMS-treated conditions are an indirect consequence of altered cellular physiology resulting from KU70 deletion. For instance, changes in the expression of DNA damage response genes, stress tolerance pathways, or chromatin organization may influence the cell’s susceptibility to mutagenic agents, thereby affecting mutation rates. These effects are likely independent of direct participation of NHEJ in lesion bypass or repair, which are primarily mediated by TLS and nucleotide/base excision pathways.

Rev1 and Pol ζ contribute to most UV- and MMS-induced mutations in Y. lipolytica

Consistent with previous findings in the model yeast S. cerevisiae (Lawrence 2002; Sale et al. 2012), we observed that deletion of REV1 or REV3 in Y. lipolytica resulted in a notable reduction in the rate of spontaneous SNVs (Fig. 5A). In contrast, deletion of RAD30 had no observable effect on the spontaneous SNV rate (Fig. 5A). More importantly, both REV1 and REV3 are essential for the majority of mutagenesis induced by MMS and UV treatment, as indicated by the dramatic suppression of SNV frequency in the rev1 and rev3 mutants (Figs. 3A and 5A). A similar result was observed in Zeocin-treated Y. lipolytica, in which the SNV rate decreased by 59% and 67% in the absence of Rev1 and Rev3, respectively (Xiong et al. 2025). These findings strongly suggest that Rev1 and Pol ζ are major contributors to DNA damage-induced mutagenesis in Y. lipolytica. Therefore, although TLS promotes cell survival under genotoxic stress (Fig. S1) (Zheng et al. 2022), it does so at the cost of increased mutagenesis, a tradeoff that appears conserved in Y. lipolytica.

As a Y-family DNA polymerase, yeast Rev1 possesses deoxycytidyl transferase activity and primarily functions as a scaffolding protein that recruits and coordinates other TLS polymerases—most notably DNA Pol ζ—to stalled replication forks (Haracska et al. 2002; Auerbach and Demple 2010). Rev1 and Pol ζ cooperate in two distinct models to explain their roles in damage-induced mutagenesis. In the first model, Rev1 directly participates in lesion bypass by inserting a dCMP opposite damaged bases, particularly abasic sites (Kim et al. 2011; Zheng et al. 2022), thereby initiating mutagenic translesion synthesis. In the second model, Rev1 serves a non-catalytic yet essential scaffolding role, acting as a structural component of the Pol ζ complex to promote nucleotide insertion across DNA lesions. If Y. lipolytica Rev1 inserts a dCMP opposite MMS-induced N³-MeA lesions, we would expect a significant elevation in A to G substitutions. Similarly, in UV-treated cells, considering that Rev1 inserts dCMP opposite CPDs, we would anticipate an increase in T to G or C to G substitutions. However, as we did not observe these expected outcomes, this suggests that Rev1 likely contributes to genotoxic stress–induced mutagenesis through the second model, where it plays a non-catalytic role as an essential component of Pol ζ for bypassing DNA lesions.

Multiple actions of Pol η in UV-induced DNA mutations

Pol η was previously identified as a specialized TLS polymerase that plays a critical role in bypassing DNA lesions caused by UV radiation (Choi and Pfeifer 2005). In the yeast S. cerevisiae, Pol η incorporates nucleotides opposite the CPDs without significant base-pairing errors, reducing the likelihood of mutations (Vandenberg et al. 2023). Compared to wild-type Y. lipolytica strain, Pol η–deficient yeast exposed to UV light had a 1.35-fold elevated SNV rate, indicating that in general, Pol η protects moderately against UV-induced mutations (Fig. 6A). Interestingly, deletion of RAD30 had divergent effects on specific base substitution types. The frequencies of C:G to T:A and C:G to A:T substitutions—commonly associated with cytosine-containing CPDs—were elevated by 1.7- and 4.4-fold, respectively, in the rad30 mutant relative to wild-type cells (Fig. 6A). This suggests that Pol η plays a protective role at cytosine sites, possibly by facilitating accurate bypass of TC or CC dimers and preventing error-prone incorporation by other TLS polymerases. In contrast, T to C substitutions were decreased by 65% due to RAD30 deletion in the UV-treated strains (Fig. 6A). Given that Rad30 in S. cerevisiae has been shown to generate T to C substitutions when bypassing 6-4PPs in an error-prone manner (Bresson and Fuchs 2002), we hypothesize that the T to C mutations observed in UV-treated Y. lipolytica arise from a similar mechanism, whereby Pol η replicates across 6-4PPs with reduced fidelity (Fig. 6B). Overall, the role of DNA Pol η in UV-treated Y. lipolytica cells—whether it reduces or increases mutagenesis—depends on the type of UV-induced lesions present on the template strand.

In addition to demonstrating the role of the NHEJ and TLS pathways in both spontaneous and genotoxic stress–induced mutagenesis, the phenotypic evolution experiments also highlighted their involvement in the adaptability to oxidative stress (Fig. 7). Given that oxidative stress is a common physiological challenge faced by yeast cells under various unfavorable growth conditions, such as elevated temperature, osmotic pressure, and antibiotic exposure (Davidson et al. 1996; Guillouzo and Guguen-Guillouzo 2020), we speculate that TLS may also contribute to adaptive evolution under other stress conditions in Y. lipolytica. From a practical perspective, enhancing the activity of NHEJ and TLS in Y. lipolytica may facilitate the acquisition of beneficial phenotypes during adaptive laboratory evolution. This strategy could potentially increase the likelihood of isolating strains with improved traits.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contribution

KZ and DZ conceived and designed research. CY, YW, XZ, YX, YF, and RN conducted experiments and analyzed data. CY, YW, KZ, CY, and DZ wrote the manuscript. All authors read and approved the manuscript.

Funding

This study was supported by grants from the National Natural Science Foundation of Zhejiang Province, China (LZ24C010002); the National Natural Science Foundation of China (32270086, 32170078, and 32022004); and the Fundamental Research Funds for the Central Universities (226–2024-00019).

Data availability

The raw data of whole genome sequencing of Y. lipolytica isolates were deposited in SRA database with the accession number of PRJNA1237737.

Declarations

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Competing interests

The authors declare no competing interests.