Loss of function of FIGNL1, a DNA damage response gene, causes human ovarian dysgenesis.

Ovarian dysgenesis (OD), an XX disorder of sex development, presents with primary amenorrhea, hypergonadotrophic hypogonadism, and infertility. In an Ashkenazi Jewish patient with OD, whole exome sequencing identified compound heterozygous frameshifts in FIGNL1, a DNA damage response (DDR) gene: c.189del and c.1519_1523del. Chromosomal breakage was significantly increased in patient cells, both spontaneously, and following mitomycin C exposure. Transfection of DYK-tagged FIGNL1 constructs in HEK293 cells showed no detectable protein in FIGNL1c.189del and truncation with reduced expression in FIGNL1c.1519_1523del (64% of wild-type [WT], P = .003). FIGNL1 forms nuclear foci increased by phleomycin treatment (20.6 ± 1.6 vs 14.8 ± 2.4, P = .02). However, mutant constructs showed reduced DYK-FIGNL1 foci formation in non-treated cells (0.8 ± 0.9 and 5.6 ± 1.5 vs 14.8 ± 2.4 in DYK-FIGNL1WT, P < .001) and no increase with phleomycin treatment. In conclusion, FIGNL1 loss of function is a newly characterized OD gene, highlighting the DDR pathway's role in ovarian development and maintenance and suggesting chromosomal breakage as an assessment tool in XX-DSD patients.

Deciphering the mechanism of processive ssDNA digestion by the Dna2-RPA ensemble.

Single-stranded DNA (ssDNA) commonly occurs as intermediates in DNA metabolic pathways. The ssDNA binding protein, RPA, not only protects the integrity of ssDNA, but also directs the downstream factor that signals or repairs the ssDNA intermediate. However, it remains unclear how these enzymes/factors outcompete RPA to access ssDNA. Using the budding yeast Saccharomyces cerevisiae as a model system, we find that Dna2 - a key nuclease in DNA replication and repair - employs a bimodal interface to act with RPA both in cis and in trans. The cis-activity makes RPA a processive unit for Dna2-catalyzed ssDNA digestion, where RPA delivers its bound ssDNA to Dna2. On the other hand, activity in trans is mediated by an acidic patch on Dna2, which enables it to function with a sub-optimal amount of RPA, or to overcome DNA secondary structures. The trans-activity mode is not required for cell viability, but is necessary for effective double strand break (DSB) repair.

DksA and DNA double-strand break repair.

We use genetic assays to suggest that transcription-coupled repair or new origin formation in Escherichia coli involves removal of RNAP to create an RNA primer for DNA synthesis. Transcription factor DksA was shown to play a role in numerous reactions involving RNA polymerase. Some, but not all, of the activities of DksA at promoters or during transcription elongation require (p)ppGpp. In addition to its role during transcription, DksA is also involved in maintaining genome integrity. Cells lacking DksA are sensitive to multiple DNA damaging agents including UV light, ionizing radiation, mitomycin C, and nalidixic acid. Here, we focus on two recent studies addressing the importance of DksA in the repair of double-strand breaks (DSBs), one by Sivaramakrishnan et al. (Nature 550:214-218, 2017) and one originating in our laboratory, Myka et al. (Mol Microbiol 111:1382-1397. https://doi.org/10.1111/mmi.14227 , 2019). It appears that depending on the type and possibly location of DNA damage, DksA can play either a passive or an active role in DSB repair. The passive role relies on exclusion of anti-backtracking factors from the RNAP secondary channel. The exact mechanism of active DksA-mediated DNA repair is unknown. However, DksA was proposed to destabilize transcription complexes, thus clearing the way for recombination and DNA repair. Based on the requirement for DksA, both in repair of DSBs and the R-loop-dependent formation of new origins of DNA replication, we propose that DksA may allow for removal of RNAP without unwinding of the RNA:DNA hybrid, which can then be extended by a DNA polymerase. This mechanism obviates the need for RNAP backtracking to repair damaged DNA.

A new role for Escherichia coli Dam DNA methylase in prevention of aberrant chromosomal replication.

The Dam DNA methylase of Escherichia coli is required for methyl-directed mismatch repair, regulation of chromosomal DNA replication initiation from oriC (which is DnaA-dependent), and regulation of gene expression. Here, we show that Dam suppresses aberrant oriC-independent chromosomal replication (also called constitutive stable DNA replication, or cSDR). Dam deficiency conferred cSDR and, in presence of additional mutations (Δtus, rpoB*35) that facilitate retrograde replication fork progression, rescued the lethality of ΔdnaA mutants. The DinG helicase was required for rescue of ΔdnaA inviability during cSDR. Viability of ΔdnaA dam derivatives was dependent on the mismatch repair proteins, since such viability was lost upon introduction of deletions in mutS, mutH or mutL; thus generation of double strand ends (DSEs) by MutHLS action appears to be required for cSDR in the dam mutant. On the other hand, another DSE-generating agent phleomycin was unable to rescue ΔdnaA lethality in dam+ derivatives (mutS+ or ΔmutS), but it could do so in the dam ΔmutS strain. These results point to a second role for Dam deficiency in cSDR. We propose that in Dam-deficient strains, there is an increased likelihood of reverse replication restart (towards oriC) following recombinational repair of DSEs on the chromosome.

Phleomycin complex - Coordination mode and in vitro cleavage of DNA.

Phleomycin is one of the anticancer glycopeptide antibiotics which cause DNA cleavage. It is commonly used as a copper(II) complex. Therefore, it is important to study the metal ion binding process and to define the coordination mode. In this paper, we describe the acid-base properties of phleomycin and the coordination sphere of the Cu(II) cation. In the metal binding process up to five nitrogen donor atoms can be involved. Four of them in the same plane deriving from: the pyrimidine ring, secondary amine of ß-aminoalanine, imidazole and amide of the nearest peptide bond (from ß-hydroxyhistidine) and in the apical position from the α-amino functional group of ß-aminoalanine, resulting complex has a square-pyramidal geometry. Phleomycin complexes are able to induce single- and double-stranded DNA damage when they are accompanied by one-electron reductants, such as dithiothreitol, glutathione, 2-mercaptoethanol or ascorbic acid. In such conditions they produce reactive oxygen species which are responsible for DNA cleavage. The metal ion binding site is relatively close to the nucleic acid interacting moiety. This supports the hypothesis that copper ion is important in the anticancer activity which involves DNA degradation.

Electroporation of germinated conidia and young mycelium as an efficient transformation system for Acremonium chrysogenum.

Three different transformation strategies were tested and compared in an attempt to facilitate and improve the genetic transformation of Acremonium chrysogenum, the exclusive producer of the pharmaceutically relevant ß-lactam antibiotic cephalosporin C. We investigated the use of high-voltage electric pulse to transform germinated conidia and young mycelium and compared these procedures with traditional PEG-mediated protoplast transformation, using phleomycin resistance as selection marker in all cases. The effect of the field strength and capacitance on transformation frequency and cell viability was evaluated. The electroporation of germinated conidia and young mycelium was found to be appropriate for transforming A. chrysogenum with higher transformation efficiencies than those obtained with the conventional protoplast-based transformation procedures. The developed electroporation strategy is fast, simple to perform, and highly reproducible and avoids the use of chemicals toxic to cells. Electroporation of young mycelium represents an alternative method for transformation of fungal strains with reduced or no sporulation, as often occurs in laboratory-developed strains in the search for high-yielding mutants for industrial bioprocesses.

Fork pausing allows centromere DNA loop formation and kinetochore assembly.

De novo kinetochore assembly, but not template-directed assembly, is dependent on COMA, the kinetochore complex engaged in cohesin recruitment. The slowing of replication fork progression by treatment with phleomycin (PHL), hydroxyurea, or deletion of the replication fork protection protein Csm3 can activate de novo kinetochore assembly in COMA mutants. Centromere DNA looping at the site of de novo kinetochore assembly can be detected shortly after exposure to PHL. Using simulations to explore the thermodynamics of DNA loops, we propose that loop formation is disfavored during bidirectional replication fork migration. One function of replication fork stalling upon encounters with DNA damage or other blockades may be to allow time for thermal fluctuations of the DNA chain to explore numerous configurations. Biasing thermodynamics provides a mechanism to facilitate macromolecular assembly, DNA repair, and other nucleic acid transactions at the replication fork. These loop configurations are essential for sister centromere separation and kinetochore assembly in the absence of the COMA complex.

Fluorescent fusions of the N protein of phage Mu label DNA damage in living cells.

The N protein of phage Mu was indicated from studies in Escherichia coli to hold linear Mu chromosomes in a circular conformation by non-covalent association, and thus suggested potentially to bind DNA double-stranded ends. Because of its role in association with linear Mu DNA, we tested whether fluorescent-protein fusions to N might provide a useful tool for labeling DNA damage including double-strand break (DSB) ends in single cells. We compared N-GFP with a biochemically well documented DSB-end binding protein, the Gam protein of phage Mu, also fused to GFP. We find that N-GFP produced in live E. coli forms foci in response to DNA damage induced by radiomimetic drug phleomycin, indicating that it labels damaged DNA. N-GFP also labels specific DSBs created enzymatically by I-SceI double-strand endonuclease, and by X-rays, with the numbers of foci corresponding with the numbers of DSBs generated, indicating DSB labeling. However, whereas N-GFP forms about half as many foci as GamGFP with phleomycin, its labeling of I-SceI- and X-ray-induced DSBs is far less efficient than that of GamGFP. The data imply that N-GFP binds and labels DNA damage including DSBs, but may additionally label phleomycin-induced non-DSB damage, with which DSB-specific GamGFP does not interact. The data indicate that N-GFP labels DNA damage, and may be useful for general, not DSB-specific, DNA-damage detection.

Lack of superoxide dismutase in a rad51 mutant exacerbates genomic instability and oxidative stress-mediated cytotoxicity in Saccharomyces cerevisiae.

A genetic analysis of synthetic lethal interactions in yeast revealed that the mutation of SOD1, encoding an antioxidant enzyme that scavenges superoxide anion radical, impaired the growth of a set of mutants defective in homologous recombination (HR) pathway. Hence, SOD1 inhibition has been proposed as a promising approach for the selective killing of HR-deficient cancer cells. However, we show that the deletion of RAD51 and SOD1 is not synthetic lethal but displays considerably slow growth and synergistic sensitivity to both reactive oxygen species (ROS)- and DNA double-strand break (DSB)-generating drugs in the budding yeast Saccharomyces cerevisiae. The function of Sod1 in regard to Rad51 is dependent on Ccs1, a copper chaperone for Sod1. Sod1 deficiency aggravates genomic instability in conjunction with the absence of Rad51 by inducing DSBs and an elevated mutation frequency. Inversely, lack of Rad51 causes a Sod1 deficiency-derived increase of intracellular ROS levels. Taken together, our results indicate that there is a significant and specific crosstalk between two major cellular damage response pathways, ROS signaling and DSB repair, for cell survival.

Structurally distinct Mre11 domains mediate MRX functions in resection, end-tethering and DNA damage resistance.

Sae2 cooperates with the Mre11-Rad50-Xrs2 (MRX) complex to initiate resection of DNA double-strand breaks (DSBs) and to maintain the DSB ends in close proximity to allow their repair. How these diverse MRX-Sae2 functions contribute to DNA damage resistance is not known. Here, we describe mre11 alleles that suppress the hypersensitivity of sae2Δ cells to genotoxic agents. By assessing the impact of these mutations at the cellular and structural levels, we found that all the mre11 alleles that restore sae2Δ resistance to both camptothecin and phleomycin affect the Mre11 N-terminus and suppress the resection defect of sae2Δ cells by lowering MRX and Tel1 association to DSBs. As a consequence, the diminished Tel1 persistence potentiates Sgs1-Dna2 resection activity by decreasing Rad9 association to DSBs. By contrast, the mre11 mutations restoring sae2Δ resistance only to phleomycin are located in Mre11 C-terminus and bypass Sae2 function in end-tethering but not in DSB resection, possibly by destabilizing the Mre11-Rad50 open conformation. These findings unmask the existence of structurally distinct Mre11 domains that support resistance to genotoxic agents by mediating different processes.

Rgf1p (Rho1p GEF) is required for double-strand break repair in fission yeast.

Rho GTPases are conserved molecules that control cytoskeletal dynamics. These functions are expedited by Rho GEFs that stimulate the release of GDP to enable GTP binding, thereby allowing Rho proteins to initiate intracellular signaling. How Rho GEFs and Rho GTPases protect cells from DNA damage is unknown. Here, we explore the extreme sensitivity of a deletion mutation in the Rho1p exchange factor Rgf1p to the DNA break/inducing antibiotic phleomycin (Phl). The Rgf1p mutant cells are defective in reentry into the cell cycle following the induction of severe DNA damage. This phenotype correlates with the inability of rgf1Δ cells to efficiently repair fragmented chromosomes after Phl treatment. Consistent with this observation Rad11p (ssDNA binding protein, RPA), Rad52p, Rad54p and Rad51p, which facilitate strand invasion in the process of homology-directed repair (HDR), are permanently stacked in Phl-induced foci in rgf1Δ cells. These phenotypes are phenocopied by genetic inhibition of Rho1p. Our data provide evidence that Rgf1p/Rho1p activity positively controls a repair function that confers resistance against the anti-cancer drug Phl.

Development of a sufficient and effective procedure for transformation of an oleaginous yeast, Rhodosporidium toruloides DMKU3-TK16.

Rhodosporidium toruloides DMKU3-TK16 (TK16), a basidiomycetous yeast isolated in Thailand, can produce a large amount of oil corresponding to approximately 70 % of its dry cell weight. However, lack of a sufficient and efficient transformation method makes further genetic manipulation of this organism difficult. We here developed a new transformation system for R. toruloides using a lithium acetate method with the Sh ble gene as a selective marker under the control of the R. toruloides ATCC 10657 GPD1 promoter. A linear DNA fragment containing the Sh ble gene expression cassette was integrated into the genome, and its integration was confirmed by colony PCR and Southern blot. Then, we further optimized the parameters affecting the transformation efficiency, such as the amount of linear DNA, the growth phase, the incubation time in the transformation mixture, the heat shock treatment temperature, the addition of DMSO and carrier DNA, and the recovery incubation time. With the developed method, the transformation efficiency of approximately 25 transformants/µg DNA was achieved. Compared with the initial trial, transformation efficiency was enhanced 417-fold. We further demonstrated the heterologous production of EGFP in TK16 by microscopic observation and immunoblot analysis, and use the technique to disrupt the endogenous URA3 gene. The newly developed method is thus simple and time saving, making it useful for efficient introduction of an exogenous gene into R. toruloides strains. Accordingly, this new practical approach should facilitate the molecular manipulation, such as target gene introduction and deletion, of TK16 and other R. toruloides strains as a major source of biodiesel.

G9a inhibition potentiates the anti-tumour activity of DNA double-strand break inducing agents by impairing DNA repair independent of p53 status.

Cancer cells often exhibit altered epigenetic signatures that can misregulate genes involved in processes such as transcription, proliferation, apoptosis and DNA repair. As regulation of chromatin structure is crucial for DNA repair processes, and both DNA repair and epigenetic controls are deregulated in many cancers, we speculated that simultaneously targeting both might provide new opportunities for cancer therapy. Here, we describe a focused screen that profiled small-molecule inhibitors targeting epigenetic regulators in combination with DNA double-strand break (DSB) inducing agents. We identify UNC0638, a catalytic inhibitor of histone lysine N-methyl-transferase G9a, as hypersensitising tumour cells to low doses of DSB-inducing agents without affecting the growth of the non-tumorigenic cells tested. Similar effects are also observed with another, structurally distinct, G9a inhibitor A-366. We also show that small-molecule inhibition of G9a or siRNA-mediated G9a depletion induces tumour cell death under low DNA damage conditions by impairing DSB repair in a p53 independent manner. Furthermore, we establish that G9a promotes DNA non-homologous end-joining in response to DSB-inducing genotoxic stress. This study thus highlights the potential for using G9a inhibitors as anti-cancer therapeutic agents in combination with DSB-inducing chemotherapeutic drugs such as etoposide.

Physical Association of Saccharomyces cerevisiae Polo-like Kinase Cdc5 with Chromosomal Cohesin Facilitates DNA Damage Response.

At the onset of anaphase, a protease called separase breaks the link between sister chromatids by cleaving the cohesin subunit Scc1. This irreversible step in the cell cycle is promoted by degradation of the separase inhibitor, securin, and polo-like kinase (Plk) 1-dependent phosphorylation of the Scc1 subunit. Plk could recognize substrates through interaction between its phosphopeptide interaction domain, the polo-box domain, and a phosphorylated priming site in the substrate, which has been generated by a priming kinase beforehand. However, the physiological relevance of this targeting mechanism remains to be addressed for many of the Plk1 substrates. Here, we show that budding yeast Plk1, Cdc5, is pre-deposited onto cohesin engaged in cohesion on chromosome arms in G2/M phase cells. The Cdc5-cohesin association is mediated by direct interaction between the polo-box domain of Cdc5 and Scc1 phosphorylated at multiple sites in its middle region. Alanine substitutions of the possible priming phosphorylation sites (scc1-15A) impair Cdc5 association with chromosomal cohesin, but they make only a moderate impact on mitotic cell growth even in securin-deleted cells (pds1Δ), where Scc1 phosphorylation by Cdc5 is indispensable. The same scc1-15A pds1Δ double mutant, however, exhibits marked sensitivity to the DNA-damaging agent phleomycin, suggesting that the priming phosphorylation of Scc1 poses an additional layer of regulation that enables yeast cells to adapt to genotoxic environments.

Polymerases ε and ∂ repair dysfunctional telomeres facilitated by salt.

Damaged DNA can be repaired by removal and re-synthesis of up to 30 nucleotides during base or nucleotide excision repair. An important question is what happens when many more nucleotides are removed, resulting in long single-stranded DNA (ssDNA) lesions. Such lesions appear on chromosomes during telomere damage, double strand break repair or after the UV damage of stationary phase cells. Here, we show that long single-stranded lesions, formed at dysfunctional telomeres in budding yeast, are re-synthesized when cells are removed from the telomere-damaging environment. This process requires Pol32, an accessory factor of Polymerase δ. However, re-synthesis takes place even when the telomere-damaging conditions persist, in which case the accessory factors of both polymerases δ and ε are required, and surprisingly, salt. Salt added to the medium facilitates the DNA synthesis, independently of the osmotic stress responses. These results provide unexpected insights into the DNA metabolism and challenge the current view on cellular responses to telomere dysfunction.

The proteomic investigation reveals interaction of mdig protein with the machinery of DNA double-strand break repair.

To investigate how mineral dust-induced gene (mdig, also named as mina53, MINA, or NO52) promotes carcinogenesis through inducing active chromatin, we performed proteomics analyses for the interacting proteins that were co-immunoprecipitated by anti-mdig antibody from either the lung cancer cell line A549 cells or the human bronchial epithelial cell line BEAS-2B cells. On SDS-PAGE gels, three to five unique protein bands were consistently observed in the complexes pulled-down by mdig antibody, but not the control IgG. In addition to the mdig protein, several DNA repair or chromatin binding proteins, including XRCC5, XRCC6, RBBP4, CBX8, PRMT5, and TDRD, were identified in the complexes by the proteomics analyses using both Orbitrap Fusion and Orbitrap XL nanoESI-MS/MS in four independent experiments. The interaction of mdig with some of these proteins was further validated by co-immunoprecipitation using antibodies against mdig and its partner proteins, respectively. These data, thus, provide evidence suggesting that mdig accomplishes its functions on chromatin, DNA repair and cell growth through interacting with the partner proteins.

Variable NF-κB pathway responses in colon cancer cells treated with chemotherapeutic drugs.

BACKGROUND: The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway is activated in cells exposed to various stimuli, including those originating on the cell surface or in the nucleus. Activated NF-κB signaling is thought to enhance cell survival in response to these stimuli, which include chemotherapy and radiation. In the present effort, we determined which anticancer drugs preferentially activate NF-κB in colon cancer cells. METHODS: NF-κB reporter cells were established and treated with 5-fluorouracil (5-FU, DNA/RNA damaging), oxaliplatin (DNA damaging), camptothecin (CTP, topoisomerase inhibitor), phleomycin (radiomimetic), or erlotinib (EGFR inhibitor). The activation of NF-κB was assessed by immunofluorescence for p65 translocation, luciferase assays, and downstream targets of NF-κB activation (cIAP2, and Bcl-XL) were evaluated by immunoblotting, by ELISA (CXCL8 and IL-6 in culture supernatants), or by gene expression analysis. RESULTS: Colon cancer cells responded variably to different classes of therapeutic agents, and these agents initiated variable responses among different cell types. CPT activated NF-κB in SW480 colon cancer cells in a dose-dependent manner, but not in HCT116 cells that were either wild-type or deficient for p53. In SW480 colon cancer cells, NF-κB activation by CPT was accompanied by secretion of the cytokine CXCL8, but not by up-regulation of the anti-apoptotic genes, cIAP2 or Bcl-XL. On the contrary, treatment of HCT116 cells with CPT resulted in up-regulation of CXCR2, a receptor for CXCL8, without an increase in cytokine levels. In SW480 cells, NF-κB reporter activity, but not cytokine secretion, was inhibited by SM-7368, an NF-κB inhibitor. CONCLUSION: The results show that, in response to cancer therapeutic agents, NF-κB activation varies with the cellular make up and that drug-induced NF-κB activation may be functionally uncoupled from anti-apoptotic outcomes found for other stimuli. Some cancer cells in a heterogeneous tumor tissue may, under therapeutic pressure, release soluble factors that have paracrine activity on neighboring cells that express the cognate receptors.

An endogenous promoter for conditional gene expression in Acremonium chrysogenum: the xylan and xylose inducible promoter xyl1(P.).

Acremonium chrysogenum is the natural producer of the beta-lactam antibiotic cephalosporin C and therefore of significant biotechnological importance. Here we identified and characterized the xylanase-encoding xyl1 gene and demonstrate that its promoter, xyl1(P), is suitable for conditional expression of heterologous genes in A. chrysogenum. This was shown by xylose and xylan-inducible xyl1(P)-driven expression of genes encoding green fluorescence protein and phleomycin resistance. Moreover, we demonstrate the potential of the xyl1(P) promoter for selection marker recycling. Taken together, these finding will help to overcome the limitation in genetic tools in this important filamentous fungus.

Dual gene expression cassette vectors with antibiotic selection markers for engineering in Saccharomyces cerevisiae.

BACKGROUND: Manipulations in Saccharomyces cerevisiae classically depend on use of auxotrophy selection markers. There are several disadvantages to this in a microbial cell factory setting: (1) auxotrophies must first be engineered in prototrophic strains, and many industrial strains are polyploid/aneuploid prototrophs (2) available strain auxotrophies must be paired with available repair plasmids (3) remaining auxotrophies must be repaired prior to development of industrial bioprocesses. Use of dominant antibiotic resistance markers can circumvent these problems. However, there are relatively few yeast antibiotic resistance marker vectors available; furthermore, available vectors contain only one expression cassette, and it is often desirable to introduce more than one gene at a time. RESULTS: To overcome these problems, eight new shuttle vectors have been developed. The plasmids are maintained in yeast under a 2 µm ori and in E. coli by a pUC ori. They contain two yeast expression cassettes driven by either (1) the constitutive TEF1 and PGK1 promoters, or (2) the constitutive TEF1 promoter and the inducible GAL10 or HXT7 promoters. Expression strength of these promoters over a typical production time frame in glucose/galactose medium was examined, and identified the TEF1 and HXT7 promoters as preferred promoters over long term fermentations. Selection is provided by either aphA1 (conferring resistance to G418 in yeast and kanamycin/neomycin in E. coli) or ble (conferring resistance to phleomycin in both yeast and E. coli). Selection conditions for these plasmids/antibiotics in defined media were examined, and selection considerations are reviewed. In particular, medium pH has a strong effect on both G418 and phleomycin selection. CONCLUSIONS: These vectors allow manipulations in prototrophic yeast strains with expression of two gene cassettes per plasmid, and will be particularly useful for metabolic engineering applications. The vector set expands the (currently limited) selection of antibiotic marker plasmids available for use in yeast, and in addition makes available dual gene expression cassettes on individual plasmids using antibiotic selection. The resistance gene cassettes are flanked by loxP recognition sites to allow CreA-mediated marker removal and recycling, providing the potential for genomic integration of multiple genes. Guidelines for selection using G418 and phleomycin are provided.

A role for Myh1 in DNA repair after treatment with strand-breaking and crosslinking chemotherapeutic agents.

The highly conserved DNA glycosylase MutY is implicated in repair of oxidative DNA damage, in particular in removing adenines misincorporated opposite 7,8-dihydro-8-oxoguanine (8-oxo-G). The MutY homologues (MutYH) physically associate with proteins implicated in replication, DNA repair, and checkpoint signaling, specifically with the DNA damage sensor complex 9-1-1 proteins. Here, we ask whether MutYH could have a broader function in sensing and repairing different types of DNA damage induced by conventional chemotherapeutics. Thus, we examined if deletion of the Schizosaccharomyces pombe MutY homologue, Myh1, alone or in combination with deletion of either component of the 9-1-1 sensor complex, influences survival after exposure to different classes of DNA damaging chemotherapeutics that do not act primarily by causing 8-oxoG lesions. We show that Myh1 contributes to survival on genotoxic stresses induced by the oxidizing, DNA double strand break-inducing, bleomycins, or the DNA crosslinking platinum compounds, particularly in a rad1 mutant background. Exposure of cells to cisplatin leads to a moderate overall accumulation of Myh1 protein. Interestingly, we found that DNA damage induced by phleomycin results in increased chromatin association of Myh1. Further, we demonstrate that Myh1 relocalizes to the nucleus after exposure to hydrogen peroxide or chemotherapeutics, most prominently seen after phleomycin treatment. These observations indicate a wider role of Myh1 in DNA repair and DNA damage-induced checkpoint activation than previously thought.