Lsm12 Mediates Deubiquitination of DNA Polymerase η To Help Saccharomyces cerevisiae Resist Oxidative Stress

Abstract

In Saccharomyces cerevisiae, the Y family DNA polymerase η (Polη) regulates genome stability in response to different forms of environmental stress by translesion DNA synthesis. To elucidate the role of Polη in oxidative stress-induced DNA damage, we deleted or overexpressed the corresponding gene RAD30 and used transcriptome analysis to screen the potential genes associated with RAD30 to respond to DNA damage. Under 2 mM H₂O₂ treatment, the deletion of RAD30 resulted in a 2.2-fold decrease in survival and a 2.8-fold increase in DNA damage, whereas overexpression of RAD30 increased survival and decreased DNA damage by 1.2- and 1.4-fold, respectively, compared with the wild-type strain. Transcriptome and phenotypic analyses identified Lsm12 as a main factor involved in oxidative stress-induced DNA damage. Deleting LSM12 caused growth defects, while its overexpression enhanced cell growth under 2 mM H₂O₂ treatment. This effect was due to the physical interaction of Lsm12 with the UBZ domain of Polη to enhance Polη deubiquitination through Ubp3 and consequently promote Polη recruitment. Overall, these findings demonstrate that Lsm12 is a novel regulator mediating Polη deubiquitination to promote its recruitment under oxidative stress. Furthermore, this study provides a potential strategy to maintain the genome stability of industrial strains during fermentation.IMPORTANCE Polη was shown to be critical for cell growth in the yeast Saccharomyces cerevisiae, and deletion of its corresponding gene RAD30 caused a severe growth defect under exposure to oxidative stress with 2 mM H₂O₂ Furthermore, we found that Lsm12 physically interacts with Polη and promotes Polη deubiquitination and recruitment. Overall, these findings indicate Lsm12 is a novel regulator mediating Polη deubiquitination that regulates its recruitment in response to DNA damage induced by oxidative stress.

Keywords: DNA damage; Polη; deubiquitination; oxidative stress; recruitment.

FIG 1

RAD30 is required for S. cerevisiae growth in the presence of H₂O₂. (A) Wild-type, rad30Δ mutant, and rad30Δ/RAD30 strains were spotted on YNB plates under normal and 2 mM H₂O₂ treatment conditions. (B) The survival rates of wild-type, rad30Δ mutant, and rad30Δ/RAD30 cells over a range of H₂O₂ doses (0, 500, 1,000, 1,500, and 2,000 µM). (C) Comet assay in wild-type, rad30Δ mutant, and rad30Δ/RAD30 strains exposed to normal or 2 mM H₂O₂ conditions. Data represent the means of three biological replicates (n = 3), and error bars represent the standard deviation. *, P ≤ 0.05; **, P ≤ 0.01.

FIG 2

LSM12 is involved in DNA damage tolerance. (A) Venn diagrams depicting the numbers of upregulated and downregulated genes in wild-type and rad30Δ mutant strains under normal conditions compared with the gene expression levels in the corresponding strains under the 2 mM H₂O₂ treatment conditions. (B) Numbers of upregulated and downregulated genes in the rad30Δ mutant relative to their expression in the wild-type strain under normal and 2 mM H₂O₂ treatment conditions. (C and D) Quantitative reverse transcription-PCR (qRT-PCR) verified the mRNA expression levels of the most commonly downregulated genes, calculated relative to the ACT1 level, under normal and 2 mM H₂O₂ treatment conditions. Data represent the means of three biological replicates (n = 3), and error bars represent the standard deviation. **, P ≤ 0.01. (E) The most commonly downregulated genes were deleted, and the mutant strains were spotted on YNB plates under normal and 2 mM H₂O₂ treatment conditions. (F) The most commonly downregulated genes were overexpressed, and the mutant strains were spotted on YNB plates under normal and 2 mM H₂O₂ treatment conditions.

FIG 3

Polη interacts with Lsm12 through the UBZ domain. (A) Polη and Lsm12 were fused with the eGFP reporter and overexpressed, and the subcellular localization was visualized under normal and 2 mM H₂O₂ treatment conditions. (B) The wild-type and lsm12Δ, rad30Δ, and rad30Δ lsm12Δ mutant strains were spotted on YNB plates with or without H₂O₂. (C) Yeast two-hybrid assays confirmed the interaction between Polη and Lsm12; the D570A mutant failed to interact with Lsm12. (D) Coimmunoprecipitation assay to detect the interaction between Polη and Lsm12 in vivo. IP, immunoprecipitation.

FIG 4

Lsm12 promotes Polη focus formation. (A) Formation of Polη foci when cells of wild-type and lsm12Δ mutant strains were treated with different DNA-damaging agents. (B) Percentage of cells of different strains displaying Polη-eGFP foci in different environments. The histograms represent the mean ± standard deviation from three independent experiments. **, P ≤ 0.01.

FIG 5

Lsm12 promoted Polη deubiquitination through Ubp3. (A) The level of monoubiquitinated PCNA in the wild-type and lsm12Δ mutant strains. (B to D) The level of monoubiquitinated Polη in the wild-type strain and lsm12 mutant (B), ubp2Δ, ubp3Δ, and ubp15Δ mutant (C), and ubp3Δ, lsm12Δ, and ubp3Δ lsm12Δ mutant (D) strains. β-Actin was used as a loading control. Data represent means of three biological replicates (n = 3), and error bars represent the standard deviation. **, P ≤ 0.01. (E) Spot assays in the wild-type and ubp3Δ, lsm12Δ, and ubp3Δ lsm12Δ mutant strains with or without H₂O₂. (F) Yeast two-hybrid assays confirmed the interaction between Lsm12 and Ubp3. (G) Coimmunoprecipitation assay to detect the interaction between Lsm12 and Ubp3 in vivo.

FIG 6

Model depicting the molecular function of Lsm12. When cells are under DNA replication stress, Lsm12 binds with Ubp3 and promotes the deubiquitination of Polη, which activates the TLS pathway. In the absence of Lsm12, cells fail to deubiquitinate Polη, causing defective TLS. DDR, DNA damage response.