Previously uncharacterized genes in the UV- and MMS-induced DNA damage response in yeast

Abstract

A competitive growth assay has been used to identify yeast genes involved in the repair of UV-or MMS-induced DNA damage. A collection of 2,827 yeast strains was analyzed in which each strain has a single ORF replaced with a cassette containing two unique sequence tags, allowing for its detection by hybridization to a high-density oligonucleotide array. The hybridization data identify a high percentage of the deletion strains present in the collection that were previously characterized as being sensitive to the DNAdamaging agents. The assay, and subsequent analysis, has been used to identify six genes not formerly known to be involved in the damage response, whose deletion renders the yeast sensitive to UV or MMS treatment. The recently identified genes include three uncharacterized ORFs, as well as genes that encode protein products implicated in ubiquitination, gene silencing, and transport across the mitochondrial membrane. Epistatsis analysis of four of the genes was performed to determine the DNA damage repair pathways in which the protein products function. DNA is a labile molecule that can undergo spontaneous hydrolysis or modification by physical and chemical agents within the cellular environment (1). Modified DNA must be rapidly recognized and efficiently repaired, thus both prokaryotes and eukaryotes have evolved complex surveillance and repair mechanisms. The response to DNA damage has been well characterized in Saccharomyces cerevisiae through the isolation of mutants that are hypersensitive to specific DNA damaging agents (1). These mutation studies led to the definition of three groups of proteins involved in different types of DNA repair, termed the RAD3, RAD52, and RAD6 epistasis groups, based on phenotypic sensitivity to UV, ionizing radiation, or both.The cellular DNA damage response depends on the type of damage incurred. The UV response is largely mediated by two epistasis groups, the RAD3 group, which includes genes of the nucleotide excision and repair pathway, and the RAD6 group, encoding proteins involved in postreplication repair and damage-induced mutagenesis. Treating cells with the methylating agent methyl methanesulfonate (MMS) results in alkylated DNA, which is poorly replicated by DNA polymerases in vitro and in vivo (1, 2), and must be efficiently repaired. Base excision repair proteins (3), as well as proteins encoded by the MEC1 and RAD53 genes (2), and the RAD6 and the RAD52 epistasis group genes (4), have all been implicated in the response to MMS damage. Although excision repair and recombination repair pathways are relatively well understood, much less is known about the RAD6 mediated pathway. The response to both UV irradiation and MMS methylation likely involve additional genes, especially of the RAD6 pathway, which remain to be identified.Toward this end, we have used a collection of S. cerevisiae deletion strains, wherein each strain has had an individual gene replaced by a unique DNA sequence, to phenotypically screen the yeast genome for proteins involved in the DNA da...

A Novel, Evolutionarily Conserved Protein Phosphatase Complex Involved in Cisplatin Sensitivity

“…The PP4cs complex is evolutionarily conserved and in yeast is composed of the Pph3, Ybl046w, and Psy2 proteins. The conservation in S. cerevisiae suggested a role for PP4cs in resistance to cytotoxic agents: genome-wide screens of the yeast deletion collection with the DNA-damaging agents cisplatin, oxaliplatin, mitomycin C, and methyl methanesulfate have highlighted the importance of both PPH3 and PSY2 in survival following exposure to these agents (10,38). We demonstrated that yeast strains deleted for YBL046W (PP4R2) are hypersensitive to cisplatin to the same extent as strains deleted for either PPH3 or PSY2.…”

“…Interestingly PPH3 deletion was also found to render cells hypersensitive to cisplatin treatment (10). Deletion of PPH3 or PSY2 was also reported to moderately increase the sensitivity of S. cerevisiae to methyl methanesulfonate (38). Because in mammalian cells PP4R2 is an obligatory partner for the association of PP4C and PP4R3, and Tip41 associates with PP4cs in yeast and mammals, we characterized the sensitivity of yeast lacking the YBL046W or TIP41 (YPR040W) genes to cisplatin treatment.…”

Role of Doa1 in the Saccharomyces cerevisiae DNA Damage Response

“…Other studies showed that Doa1 binds proteins containing UBX (Ub regulatory X) domains, such as Ubx4, Ubx6, and Ubx7 (7,43). Doa1 was first implicated as being part of the DNA damage response in a genome-wide screen for genes involved in repairing UV-and MMS-induced DNA damage (17). More recently, it was shown that LUB1, the Schizosaccharomyces pombe homologue of DOA1, plays a role in mediating the stress response (30).…”

Novel functions of the phosphatidylinositol metabolic pathway discovered by a chemical genomics screen with wortmannin

“…A chemical genomics screen for rapamycin sensitivity with deletion strains covering one-third of the yeast genome has previously been reported (6); this study explored the broad functions of the rapamycin-sensitive Tor pathways. In addition, screens of the yeast diploid deletion set have uncovered novel genes involved in resistance to ionizing radiation, UV, and methyl methanesulfonate (MMS)-induced DNA damage (7)(8)(9). Here, we report a whole-genome screen for altered sensitivity to wortmannin, a small-molecule modulator of the phosphatidylinositol (PI) metabolic pathway.…”