Yeast and human genes that affect the
            <i>Escherichia coli</i>
            SOS response

Perkins, Edward L.; Sterling, Joan F.; Hashem, Vera I.; Resnick, Michael A.

doi:10.1073/pnas.96.5.2204

Cited by 25 publications

(13 citation statements)

References 52 publications

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“…The PC4 gene was isolated by transforming the E. coli fpg mutY strain with a human cDNA library constructed in a bacterial expression vector (45,54) and then screening individual transformed colonies for suppression of the spontaneous mutator phenotype. PC4 is one of the strong antimutators identified (Fig.…”

Section: Resultsmentioning

confidence: 99%

The Single-Strand DNA Binding Activity of Human PC4 Prevents Mutagenesis and Killing by Oxidative DNA Damage

Wang

Sarker

Cooper

et al. 2004

Molecular and Cellular Biology

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Human positive cofactor 4 (PC4) is a transcriptional coactivator with a highly conserved single-strand DNA (ssDNA) binding domain of unknown function. We identified PC4 as a suppressor of the oxidative mutator phenotype of the Escherichia coli fpg mutY mutant and demonstrate that this suppression requires its ssDNA binding activity. Saccharomyces cerevisiae mutants lacking their PC4 ortholog Sub1 are sensitive to hydrogen peroxide and exhibit spontaneous and peroxide-induced hypermutability. PC4 expression suppresses the peroxide sensitivity of the yeast sub1⌬ mutant, suggesting that the human protein has a similar function. A role for yeast and human proteins in DNA repair is suggested by the demonstration that Sub1 acts in a peroxide resistance pathway involving Rad2 and by the physical interaction of PC4 with the human Rad2 homolog XPG. We show that XPG recruits PC4 to a bubble-containing DNA substrate with a resulting displacement of XPG and formation of a PC4-DNA complex. We discuss the possible requirement for PC4 in either global or transcription-coupled repair of oxidative DNA damage to mediate the release of XPG bound to its substrate.Oxidative DNA damage and the mutations it causes have been implicated in a number of human diseases, including cancer and neurodegenerative diseases, and are a contributing factor to aging (6,12,35,36). Thus, a thorough understanding of genes involved in the prevention and repair of oxidative DNA damage and its mutagenic consequences is important to our understanding of the mechanisms mitigating these diseases and exacerbating normal degenerative processes associated with aging.Oxidative DNA damage results from the interaction of reactive oxygen species (ROS) with DNA. ROS are produced as by-products of normal aerobic metabolism and by exogenous factors, such as ionizing radiation and chemical oxidants (6,12,35,36). The deleterious consequences of ROS to an organism's genetic material are held in check by proteins that prevent or repair oxidative DNA damage (7,12,13,15,21). Unrepaired oxidative lesions result in increased mutagenesis, lethality, and apoptosis (23,27). A balance between DNA repair and damage prevention mechanisms and ROS production is required to maintain a low spontaneous mutation rate. Factors that increase ROS production, reduce ROS detoxification, or factors that affect repair of oxidative DNA lesions result in increased mutagenesis. This is best demonstrated in Escherichia coli; mutations that inactivate the fpg and mutY genes, whose products repair the predominant oxidative lesion 8-oxoguanine (8-oxoG) and its mispaired intermediate 8-oxoG:A, respectively, result in a mutator phenotype that specifically increases GC3 TA transversion mutagenesis (37).In this study, we screened a human cDNA library and describe the isolation and characterization of the human transcription positive cofactor 4 (PC4) gene as a suppressor of oxidative mutagenesis in the E. coli fpg mutY strain. We demonstrate that PC4 and its Saccharomyces cerevisiae ortholog SUB1 are requi...

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Section: Resultsmentioning

confidence: 99%

The Single-Strand DNA Binding Activity of Human PC4 Prevents Mutagenesis and Killing by Oxidative DNA Damage

Wang

Sarker

Cooper

et al. 2004

Molecular and Cellular Biology

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“…This protein product is predicted to be 332 aa in length and 30% identical over 130 aa to the Lin-1p of Caenorhabditis elegans, which is thought to regulate cell cycle progression (31). When overexpressed in Escherichia coli, Ypl055p was found to induce SOS in a RecAdependent fashion (32). Recently, Resnick and colleagues (33) identified ypl055c⌬ as sensitive to ␥ irradiation.…”

Section: Discussionmentioning

confidence: 99%

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

Hanway

Chin

Xia

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

102

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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...

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“…The use of spontaneous oxidation as the DNA damaging treatment provides a high degree of sensitivity compared with methods used by others (18,29), because mutagenic oxidative damage is constantly occurring, thereby allowing mutations to accumulate in these sensitive strains of E. coli. Expression of cDNAs that result in either a small reduction in the production of DNA damage or a small increase in DNA repair activity reduces the number of spontaneous mutations.…”

Section: Discussionmentioning

confidence: 99%

“…Competent MV3884 cells were transformed with 600 ng of cDNA present in the pSE380 vector, which contains an isopropyl-␤-D-thiogalactoside (IPTG)-inducible synthetic promoter that functions in E. coli (18). Transformants were selected on LB ampicillin plates.…”

Section: Methodsmentioning

confidence: 99%

Functional genomics reveals a family of eukaryotic oxidation protection genes

Volkert

Elliott

Housman

2000

Proc. Natl. Acad. Sci. U.S.A.

105

114

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Reactive oxygen species (ROS) are toxic compounds produced by normal metabolic processes. Their reactivity with cellular components is a major stress for aerobic cells that results in lipid, protein, and DNA damage. ROS-mediated DNA damage contributes to spontaneous mutagenesis, and cells deficient in repair and protective mechanisms have elevated levels of spontaneous mutations. In Escherichia coli a large number of genes are involved in the repair of oxidative DNA damage and its prevention by detoxification of ROS. In humans, the genes required for these processes are not well defined. In this report we describe the human OXR1 (oxidation resistance) gene discovered in a search for human genes that function in protection against oxidative damage. OXR1 is a member of a conserved family of genes found in eukaryotes but not in prokaryotes. We also outline the procedures developed to identify human genes involved in the prevention and repair of oxidative damage that were used to identify the human OXR1 gene. This procedure makes use of the spontaneous mutator phenotype of E. coli oxidative repair-deficient mutants and identifies genes of interest by screening for antimutator activity resulting from cDNA expression.

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Yeast and human genes that affect the Escherichia coli SOS response

Cited by 25 publications

References 52 publications

The Single-Strand DNA Binding Activity of Human PC4 Prevents Mutagenesis and Killing by Oxidative DNA Damage

The Single-Strand DNA Binding Activity of Human PC4 Prevents Mutagenesis and Killing by Oxidative DNA Damage

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

Functional genomics reveals a family of eukaryotic oxidation protection genes

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