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E C Friedberg

Publications and source records attributed to E C Friedberg.

At least 109 records · Page 6Linked to original sources
PubMed1993

RAD9-dependent G1 arrest defines a second checkpoint for damaged DNA in the cell cycle of Saccharomyces cerevisiae.

Exposure of the yeast Saccharomyces cerevisiae to ultraviolet (UV) light, the UV-mimetic chemical 4-nitroquinoline-1-oxide (4NQO), or gamma radiation after release from G1 arrest induced by alpha factor results in delayed resumption of the cell cycle. As is the case with G2 arrest following ionizing radiation damage [Weinert, T. A. & Hartwell, L. H. (1988) Science 241, 317-322], the normal execution of DNA damage-induced G1 arrest depends on a functional yeast RAD9 gene. We suggest that the RAD9 gene product may interact with cellular components common to the G1/S and G2/M transition points in the cell cycle of this yeast. These observations define a checkpoint in the eukaryotic cell cycle that may facilitate the repair of lesions that are otherwise processed to lethal and/or mutagenic damage during DNA replication. This checkpoint apparently operates after the mating pheromone-induced G1 arrest point but prior to replicative DNA synthesis, S phase-associated maximal induction of histone H2A mRNA, and bud emergence.

Base Sequence↗
PubMed1993

Nucleotide-excision repair of DNA in cell-free extracts of the yeast Saccharomyces cerevisiae.

A wide spectrum of DNA lesions are repaired by the nucleotide-excision repair (NER) pathway in both eukaryotic and prokaryotic cells. We have developed a cell-free system in Saccharomyces cerevisiae that supports NER. NER was monitored by measuring repair synthesis in DNA treated with cisplatin or with UV radiation. Repair synthesis in vitro was defective in extracts of rad1, rad2, and rad10 mutant cells, all of which have mutations in genes whose products are known to be required for NER in vivo. Additionally, repair synthesis was complemented by mixing different mutant extracts, or by adding purified Rad1 or Rad10 protein to rad1 or rad10 mutant extracts, respectively. The latter observation demonstrates that the Rad1 and Rad10 proteins directly participate in the biochemical pathway of NER. NER supported by nuclear extracts requires ATP and Mg2+ and is stimulated by polyethylene glycol and by small amounts of whole cell extract containing overexpressed Rad2 protein. The nuclear extracts also contain base-excision repair activity that is present at wild-type levels in rad mutant extracts. This cell-free system is expected to facilitate studies on the biochemical pathway of NER in S. cerevisiae.

Cell Nucleus↗
PubMed1993

The DNA helicase activities of Rad3 protein of Saccharomyces cerevisiae and helicase II of Escherichia coli are differentially inhibited by covalent and noncovalent DNA modifications.

Rad3 protein of Saccharomyces cerevisiae is a DNA-dependent ATPase that acts as a DNA helicase on partially duplex substrates. Rad3 protein is required for damage-specific incision of DNA during the nucleotide excision repair (NER) pathway in yeast. Helicase II of Escherichia coli is also a DNA helicase, but it is involved in postincision events in NER. Previous investigations have demonstrated that the DNA helicase activities of Rad3 protein and helicase II are both inhibited by DNA damage. In the present study we have compared the response of yeast Rad3 protein and E. coli helicase II to a broad spectrum of DNA modifications. The Rad3 helicase activity is considerably more sensitive to ultraviolet radiation damage and cisplatin adducts in DNA than to drugs that interact noncovalently with duplex DNA. Conversely, E. coli helicase II is highly sensitive to noncovalent DNA modifications but less sensitive than Rad3 protein to ultraviolet radiation damage or cisplatin adducts. We also show that Rad3 protein and helicase II differ in their ability to form stable protein-DNA complexes at sites of DNA damage. Hence, DNA helicases that catalyze distinct steps in NER respond differently to chemical and conformational states of the DNA substrate. The observation that Rad3 protein is particularly sensitive to covalent but not noncovalent alterations in DNA structure is consistent with the hypothesis that this enzyme may have adopted a highly specialized role in damage-specific recognition during NER.

Adenosine Triphosphatases↗
PubMed1993

Yeast DNA repair and recombination proteins Rad1 and Rad10 constitute a single-stranded-DNA endonuclease.

Damage-specific recognition and incision of DNA during nucleotide excision repair in yeast and mammalian cells requires multiple gene products. Amino-acid sequence homology between several yeast and mammalian genes suggests that the mechanism of nucleotide excision repair is conserved in eukaryotes, but very little is known about its biochemistry. In the yeast Saccharomyces cerevisiae at least 6 genes are needed for this process, including RAD1 and RAD10 (ref. 1). Mutations in the two genes inactivate nucleotide excision repair and result in a reduced efficiency of mitotic recombinational events between repeated sequences. The Rad10 protein has a stable and specific interaction with Rad1 protein and also binds to single-stranded DNA and promotes annealing of homologous single-stranded DNA. The amino-acid sequence of the yeast Rad10 protein is homologous with that of the human excision repair gene ERCC1 (ref. 3). Here we demonstrate that a complex of purified Rad1 and Rad10 proteins specifically degrades single-stranded DNA by an endonucleolytic mechanism. This endonuclease activity is presumably required to remove non-homologous regions of single-stranded DNA during mitotic recombination between repeated sequences as previously suggested, and may also be responsible for the specific incision of damaged DNA during nucleotide excision repair.

Bacteriophages↗
PubMed1993

Inhibition of Rad3 DNA helicase activity by DNA adducts and abasic sites: implications for the role of a DNA helicase in damage-specific incision of DNA.

The yeast nucleotide excision repair gene RAD3 is absolutely required for damage-specific incision of DNA. Rad3 protein is a DNA helicase, and previous studies have shown that its catalytic activity is inhibited by ultraviolet (UV) radiation damage. This inhibition is observed when base damage is confined to the DNA strand on which Rad3 protein binds and translocates, and not when damage is present exclusively on the complementary strand. In the present study, we show that Rad3 DNA helicase activity is inhibited in an identical strand-specific fashion by bulky base adducts formed by treating DNA with the antineoplastic agent cisplatin or the antibiotic compound CC-1065, which alter the secondary structure of DNA in different ways. In addition, Rad3 helicase activity is inhibited by small adducts generated by treatment of DNA with diethyl sulfate and by the presence of sites at which pyrimidines have been lost (abasic sites). No inhibition of Rad3 helicase activity was detected when DNA was methylated at various base positions. Cisplatin-modified single-stranded DNA and poly(deoxyuridylic acid) containing abasic sites are more effective competitors for Rad3 helicase activity than their undamaged counterparts, suggesting that Rad3 protein is sequestered at such lesions, resulting in the formation of stable Rad3 protein-DNA complexes. The observations of strand-specific inhibition of Rad3 helicase activity and the formation of stable complexes with the covalently modified strand suggest a general mechanism by which the RAD3 gene product may be involved in nucleotide excision repair in yeast.

Adenine↗
PubMed1993

Yeast DNA recombination and repair proteins Rad1 and Rad10 constitute a complex in vivo mediated by localized hydrophobic domains.

The Saccharomyces cerevisiae Rad1 and Rad10 proteins are required for damage-specific incision during nucleotide excision repair and also for certain mitotic recombination events between repeated sequences. Previously we have demonstrated that Rad1 and Rad10 form a specific complex in vitro. Using the 'two-hybrid' genetic assay system we now report that Rad1 and Rad10 proteins are subunits of a specific complex in the cell nucleus. The Rad10-binding domain of Rad1 protein maps to a localized region between amino acids 809-997. The Rad1-binding domain of Rad10 protein maps between amino acids 90-210. These domains are evolutionarily conserved and are hydrophobic in character. Although significant homology exists between Rad10 and the human-DNA-repair protein Ercc1 in this region, we were unable to detect any interaction between Ercc1 and Rad1 proteins. We conclude that Rad1 and Rad10 operate in DNA repair and mitotic recombination as a constitutive complex.

Base Sequence↗
PubMed1993

Evidence that the Rad1 and Rad10 proteins of Saccharomyces cerevisiae participate as a complex in nucleotide excision repair of UV radiation damage.

A newly characterized rad1 missense mutation (rad1-20) in the yeast Saccharomyces cerevisiae maps to a region of the Rad1 polypeptide known to be required for Rad1-Rad10 complex formation. The UV sensitivity of the rad1-20 mutant can be partially and specifically corrected by overexpression of wild-type Rad10 protein. These results suggest that complex formation between the Rad1 and Rad10 proteins is required for nucleotide excision repair.

Amino Acid Sequence↗
PubMed1993

DNA repair synthesis during base excision repair in vitro is catalyzed by DNA polymerase epsilon and is influenced by DNA polymerases alpha and delta in Saccharomyces cerevisiae.

Base excision repair is an important mechanism for correcting DNA damage produced by many physical and chemical agents. We have examined the effects of the REV3 gene and the DNA polymerase genes POL1, POL2, and POL3 of Saccharomyces cerevisiae on DNA repair synthesis is nuclear extracts. Deletional inactivation of REV3 did not affect repair synthesis in the base excision repair pathway. Repair synthesis in nuclear extracts of pol1, pol2, and pol3 temperature-sensitive mutants was normal at permissive temperatures. However, repair synthesis in pol2 nuclear extracts was defective at the restrictive temperature of 37 degrees C and could be complemented by the addition of purified yeast DNA polymerase epsilon. Repair synthesis in pol1 nuclear extracts was proficient at the restrictive temperature unless DNA polymerase alpha was inactivated prior to the initiation of DNA repair. Thermal inactivation of DNA polymerase delta in pol3 nuclear extracts enhanced DNA repair synthesis approximately 2-fold, an effect which could be specifically reversed by the addition of purified yeast DNA polymerase delta to the extract. These results demonstrate that DNA repair synthesis in the yeast base excision repair pathway is catalyzed by DNA polymerase epsilon but is apparently modulated by the presence of DNA polymerases alpha and delta.

Base Sequence↗
PubMed1992

DNA ligase I from Saccharomyces cerevisiae: physical and biochemical characterization of the CDC9 gene product.

Genetic studies have previously demonstrated that the Saccharomyces cerevisiae CDC9 gene product, which is functionally homologous to mammalian DNA ligase I, is required for DNA replication and is also involved in DNA repair and genetic recombination. In the present study we have purified the yeast enzyme. When measured under denaturing conditions, Cdc9 protein has a polypeptide molecular mass of 87 kDa. The native form of the enzyme is an 80-kDa asymmetric monomer. Both estimates are in good agreement with the M(r) = 84,406 predicted from the translated sequence of the CDC9 gene. Cdc9 DNA ligase acts via the same basic reaction mechanism employed by all known ATP-dependent DNA ligases. The catalytic functions reside in a 70-kDa C-terminal domain that is conserved in mammalian DNA ligase I and in Cdc17 DNA ligase from Schizosaccharomyces pombe. The ATP analog ATP alpha S inhibits the ligation reaction, although Cdc9 protein does form an enzyme-thioadenylate intermediate. Since Cdc9 DNA ligase exhibited the same substrate specificity as mammalian DNA ligase I, this enzyme can be considered to be the DNA ligase I of S. cerevisiae. There is genetic evidence suggesting that DNA ligase may be directly involved in error-prone DNA repair. We examined the ability of Cdc9 DNA ligase to join nicks with mismatches at the termini. Mismatches at the 5' termini of nicks had very little effect on ligation, whereas mismatches opposite a purine at 3' termini inhibited DNA ligation. The joining of DNA molecules with mismatched termini by DNA ligase may be responsible for the generation of mutations.

Adenosine Monophosphate↗
PubMed1992

A putative second adenylate kinase-encoding gene from the yeast Saccharomyces cerevisiae.

Sequencing of the region upstream from the yeast RAD3 gene has revealed an open reading frame (ORF) of 225 amino acids (aa) that could encode a 25.3-kDa polypeptide. The predicted aa sequence of this ORF is homologous with that of several eukaryotic adenylate kinase (Adk)-encoding genes, including the yeast gene, ADK1. These findings suggest that the yeast Saccharomyces cerevisiae has a second Adk-encoding gene, tentatively designated as ADK2.

Adenylate Kinase↗
PubMed1992

Excision repair of DNA in nuclear extracts from the yeast Saccharomyces cerevisiae.

Excision repair of DNA is an important cellular response to DNA damage caused by a broad spectrum of physical and chemical agents. We have established a cell-free system in which damage-specific DNA repair synthesis can be demonstrated in vitro with nuclear extracts from the yeast Saccharomyces cerevisiae. Repair synthesis of UV-irradiated plasmid DNA was observed in a radiation dose-dependent manner and was unaffected by mutations in the RAD1, RAD2, RAD3, RAD4, RAD10, or APN1 genes. DNA damaged with cis-platin was not recognized as a substrate for repair synthesis. Further examination of the repair synthesis observed with UV-irradiated DNA revealed that it is dependent on the presence of endonuclease III-sensitive lesions in DNA, but not pyrimidine dimers. These observations suggest that the repair synthesis observed in yeast nuclear extracts reflects base excision repair of DNA. Our data indicate that the patch size of this repair synthesis is at least seven nucleotides. This system is expected to facilitate the identification of specific gene products which participate in base excision repair in yeast.

Cell Nucleus↗
PubMed1992

The DNA helicase and adenosine triphosphatase activities of yeast Rad3 protein are inhibited by DNA damage. A potential mechanism for damage-specific recognition.

Purified Rad3 protein from the yeast Saccharomyces cerevisiae is a single-stranded DNA-dependent ATPase and also acts as a DNA helicase on partially duplex DNA. In this study we show that the DNA helicase activity is inhibited when a partially duplex circular DNA substrate is exposed to ultraviolet (UV) radiation. Inhibition of DNA helicase activity is sensitive to the particular strand of the duplex region which carries the damage. Inhibition is retained if the single-stranded circle is irradiated prior to annealing to an unirradiated oligonucleotide, but not if a UV-irradiated oligonucleotide is annealed to unirradiated circular single-stranded DNA. UV irradiation of single-stranded DNA or deoxyribonucleotide homopolymers also inhibits the ability of these polynucleotides to support the hydrolysis of ATP by Rad3 protein. UV radiation damage apparently blocks translocation of Rad3 protein and results in the formation of stable Rad3 protein-UV-irradiated DNA complexes. As a consequence, Rad3 protein remains sequestered on DNA, presumably at sites of base damage. The sensitivity of Rad3 protein to the presence of DNA damage on the strand along which it translocates provides a potential mechanism for damage recognition during nucleotide excision repair and may explain the absolute requirement for Rad3 protein for damage-specific incision of DNA in yeast.

Adenosine Triphosphatases↗
PubMed1992

Correction of xeroderma pigmentosum complementation group D mutant cell phenotypes by chromosome and gene transfer: involvement of the human ERCC2 DNA repair gene.

Cultured cells from individuals afflicted with the genetically heterogeneous autosomal recessive disorder xeroderma pigmentosum (XP) exhibit sensitivity to UV radiation and defective nucleotide excision repair. Complementation of these mutant phenotypes after the introduction of single human chromosomes from repair-proficient cells into XP cells has provided a means of mapping the genes involved in this disease. We now report the phenotypic correction of XP cells from genetic complementation group D (XP-D) by a single human chromosome designated Tneo. Detailed molecular characterization of Tneo revealed a rearranged structure involving human chromosomes 16 and 19, including the excision repair cross-complementing 2 (ERCC2) gene from the previously described human DNA repair gene cluster at 19q13.2-q13.3. Direct transfer of a cosmid bearing the ERCC2 gene conferred UV resistance to XP-D cells.

Blotting, Southern↗
PubMed1992

Characterization of a complex chromosomal rearrangement maps the locus for in vitro complementation of xeroderma pigmentosum group D to human chromosome band 19q13.

Microcell-mediated chromosome transfer (MMCT) is a powerful genetic technique that permits the transfer of a single chromosome from one mammalian cell to another. The utility of MMCT for gene mapping strategies is critically dependent on the careful characterization of the chromosomes being transferred. We have recently reported the identification of a single rearranged human chromosome, designated Tneo, which corrects the UV sensitivity and excision repair defect of cells of xeroderma pigmentosum genetic complementation group D (XP-D) in culture (Flejter WL et al., Proc Natl Acad Sci USA 89:261-265, 1992). Additionally, those studies demonstrated a role for the excision repair cross-complementing 2 (ERCC2) gene in the observed phenotypic correction. We now report the results of detailed conventional and molecular cytogenetic characterization of the complementing Tneo chromosome. This analysis revealed a complex rearrangement involving material from human chromosomes 16, 17, and 19. Characterization of deletions of Tneo which retained or lost XP-D complementing ability mapped the gene responsible for phenotypic correction to a small region of the terminal q-arm of this chromosome. This region includes the previously described human DNA repair gene cluster located in the region 19q13.2-q13.3, a result consistent with the notion that the in vitro correction of XP-D cells by the Tneo chromosome is rendered by the ERCC2 locus. The data illustrate the potential value of detailed cytogenetic characterization of a human chromosome present in a somatic cell hybrid, even when that material involves complex rearrangements.

Cell Line↗
PubMed1992

Regulation of the yeast RAD2 gene: DNA damage-dependent induction correlates with protein binding to regulatory sequences and their deletion influences survival.

In the yeast Saccharomyces cerevisiae the RAD2 gene is absolutely required for damage-specific incision of DNA during nucleotide excision repair and is inducible by DNA-damaging agents. In the present study we correlated sensitivity to killing by DNA-damaging agents with the deletion of previously defined specific promoter elements. Deletion of the element DRE2 increased the UV sensitivity of cells in both the G1/early S and S/G2 phases of the cell cycle as well as in stationary phase. On the other hand, increased UV sensitivity associated with deletion of the sequence-related element DRE1 was restricted to cells irradiated in G1/S. Specific binding of protein(s) to the promoter elements DRE1 and DRE2 was observed under non-inducing conditions using gel retardation assays. Exposure of cells to DNA-damaging agents resulted in increased protein binding that was dependent on de novo protein synthesis.

Base Sequence↗