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E C Friedberg

Publications and source records attributed to E C Friedberg.

At least 127 records · Page 7Linked to original sources
PubMed1992

Stable and specific association between the yeast recombination and DNA repair proteins RAD1 and RAD10 in vitro.

The RAD1 and RAD10 genes of Saccharomyces cerevisiae are two of at least seven genes which are known to be required for damage-specific recognition and/or damage-specific incision of DNA during nucleotide excision repair. RAD1 and RAD10 are also involved in a specialized mitotic recombination pathway. We have previously reported the purification of the RAD10 protein to homogeneity (L. Bardwell, H. Burtscher, W. A. Weiss, C. M. Nicolet, and E. C. Friedberg, Biochemistry 29:3119-3126, 1990). In the present studies we show that the RAD1 protein, produced by in vitro transcription and translation of the cloned gene, specifically coimmunoprecipitates with the RAD10 protein translated in vitro or purified from yeast. Conversely, in vitro-translated RAD10 protein specifically coimmunoprecipitates with the RAD1 protein. The sites of this stable and specific interaction have been mapped to the C-terminal regions of both polypeptides. This portion of RAD10 protein is evolutionarily conserved. These results are the first biochemical evidence of a specific association between any eukaryotic proteins genetically identified as belonging to a recombination or DNA repair pathway and suggest that the RAD1 and RAD10 proteins act at the same or consecutive biochemical steps in both nucleotide excision repair and mitotic recombination.

Base Sequence↗
PubMed1991

Nucleotide excision repair of DNA by human cell extracts is suppressed in reconstituted nucleosomes.

Nucleotide excision repair is a complex biochemical process that corrects DNA damage caused by a broad spectrum of physical and chemical agents. We examined the effect of the assembly of ultraviolet-irradiated plasmid DNA into nucleosomes on nucleotide excision repair supported by human cell extracts. Repair synthesis in unassembled UV-irradiated plasmid DNA was readily detected in extracts from repair-proficient human cells. In contrast, repair synthesis was markedly suppressed in UV-irradiated DNA assembled into nucleosomes (minichromosomes). This suppression occurred at a step(s) which precedes repair synthesis during nucleotide excision repair. Human cell extracts were unable to effectively assemble plasmid DNA into nucleosomes under repair synthesis conditions. The addition of purified histones to the extracts restored their capacity for nucleosome assembly and simultaneously led to the suppression of repair synthesis. We propose that the preferential repair of actively transcribed genes relative to transcriptionally silent genes may reflect altered nucleosome conformation during transcription.

Animals↗
PubMed1991

Functional complementation of ataxia-telangiectasia group D (AT-D) cells by microcell-mediated chromosome transfer and mapping of the AT-D locus to the region 11q22-23.

The hereditary human disease ataxia-telangiectasia (AT) is characterized by phenotypic complexity at the cellular level. We show that multiple mutant phenotypes of immortalized AT cells from genetic complementation group D (AT-D) are corrected after the introduction of a single human chromosome from a human-mouse hybrid line by microcell-mediated chromosome transfer. This chromosome is cytogenetically abnormal. It consists primarily of human chromosome 18, but it carries translocated material from the region 11q22-23, where one or more AT genes have been previously mapped by linkage analysis. A cytogenetically normal human chromosome 18 does not complement AT-D cells after microcell-mediated transfer, whereas a normal human chromosome 11 does. We conclude that the AT-D gene is located on chromosome 11q22-23.

Ataxia Telangiectasia↗
PubMed1991

Eukaryotic DNA repair: glimpses through the yeast Saccharomyces cerevisiae.

Eukaryotic cells are able to mount several genetically complex cellular responses to DNA damage. The yeast Saccharomyces cerevisiae is a genetically well characterized organism that is also amenable to molecular and biochemical studies. Hence, this organism has provided a useful and informative model for dissecting the biochemistry and molecular biology of DNA repair in eukaryotes.

Animals↗
PubMed1991

Yeast genes involved in DNA-repair processes: new looks on old faces.

Multiple genes (many of which are designated RAD (for radiation resistance)) are required for cellular responses to DNA damage in the yeast Saccharomyces cerevisiae. In recent years a number of these genes have been cloned and sequenced and in some cases their polypeptide products have been purified and characterized biochemically. These studies are beginning to yield clues about the possible nature of the multiple biochemical pathways for DNA-damage processing in yeast.

Adenosine Triphosphatases↗
PubMed1990

Characterization of the RAD10 gene of Saccharomyces cerevisiae and purification of Rad10 protein.

The RAD10 gene of Saccharomyces cerevisiae is one of at least five genes required for damage-specific incision of DNA during nucleotide excision repair. This gene was previously cloned and sequenced [Weiss, W. A., & Friedberg, E. C. (1985) EMBO J. 4, 1575-1582; Reynolds et al. (1985) EMBO J. 4, 3549-3552]. In the present studies, we have mapped one major and three minor transcriptional start sites in the RAD10 gene. The locations of these sites relative to the translational start codon are remarkably similar to those previously identified in the yeast RAD2 gene [Nicolet et al. (1985) Gene 36, 225-234]. The two genes also share common sequences in these regions. However, in contrast to RAD2 [Robinson et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 1842-1846], RAD10 is not induced following exposure of cells to the DNA-damaging agent 4-nitroquinoline 1-oxide. Native RAD10 protein and also two different Rad10 fusion proteins are rapidly degraded in most Escherichia coli strains. However, following overexpression of the cloned RAD10 gene in yeast, native Rad10 protein was purified to greater than 90% homogeneity. A catalytic function has not been identified for the purified protein. RAD10 cells (untransformed with the cloned gene) contain fewer than 500 molecules per cell. This is similar to the levels of the UvrA, UvrB, and UvrC nucleotide excision repair proteins in E. coli.

4-Nitroquinoline-1-oxide↗
PubMed1990

Gene complementing xeroderma pigmentosum group A cells maps to distal human chromosome 9q.

Phenotypic complementation of xeroderma pigmentosum group A (XP-A) cells by microcell-mediated transfer of a single rearranged neo-tagged human chromosome from a human-mouse somatic cell hybrid designated K3SUB1A9-3 was reported previously. Extended growth of this human-mouse hybrid in culture led to deletion of the small arm of the human chromosome, with concomitant loss of complementing ability when introduced into XP-A cells by microcell-mediated chromosome transfer. Cytogenetic analysis of both hybrids suggests that the complementing locus is on chromosome 9q22.2-q34.3, and Southern blot analysis confirms the presence of distal chromosome 9q sequences.

Animals↗
PubMed1990

Influence of DNA repair deficiencies on the UV sensitivity of yeast cells in different cell cycle stages.

Synchronously dividing haploid yeast cells were UV-irradiated in various stages of the cell cycle after release from alpha-factor arrest. In confirmation of earlier results (Chanet et al., 1973), in wild-type strains G1/S phase cells were found to be the most sensitive and late S/G2 cells the most resistant. Stationary-phase (G0) cells were significantly more UV resistant than G1 cells. Strains defective in nucleotide excision repair lost enhanced resistance in the G2 phase and were most UV-sensitive in the G0 state. Reduced G2 resistance was also observed in rad6 mutants but not in rad9 mutants. After UV-irradiation in G1 phase rad9 mutant cells showed a reduced G1/S phase arrest.

Cell Cycle↗
PubMed1990

Effects of multiple yeast rad3 mutant alleles on UV sensitivity, mutability, and mitotic recombination.

A yeast strain was constructed that had a disruption of the chromosomal RAD3 gene and carried a series of centromeric plasmids with defined mutations in this gene. Using this isogenic collection, we examined sensitivity to UV radiation, spontaneous and UV radiation-induced mutagenesis, and mitotic recombination. Several alleles resulted in a marked increase in UV sensitivity. Most of these alleles were found to carry mutations located in consensus motifs for DNA helicases. Other alleles caused a modest or no increase in UV sensitivity and carried mutations in regions of the Rad3 polypeptide that are apparently not conserved. This correlation suggests that the DNA helicase activity of Rad3 protein is required for nucleotide excision repair of DNA. Some rad3 alleles conferred a marked increase in the frequency of spontaneous mutagenesis, including nonsuppressor reversion of the lys2-1 ochre mutation. These alleles also showed a good correlation with conserved DNA helicase domains, suggesting that the Rad3 DNA helicase also plays a role in the fidelity of DNA synthesis or postreplicative mismatch correction. Several rad3 mutator alleles also resulted in increased levels of mitotic recombination. Increased spontaneous mutagenesis and mitotic recombination are characteristic features of the Rem- phenotype. However, in contrast to the prototypic Rem- phenotype, the rad3 mutator alleles identified in this study did not confer inviability in the presence of mutations in the RAD50 or RAD52 gene required for strand break repair of DNA.

Alleles↗
PubMed1989

Purification and characterization of Rad3 ATPase/DNA helicase from Saccharomyces cerevisiae.

The Rad3 ATPase/DNA helicase was purified to physical homogeneity from extracts of yeast cells containing overexpressed Rad3 protein. The DNA helicase can unwind duplex regions as short as 11 base pairs in a partially duplex circular DNA substrate and does so by a strictly processive mechanism. On partially duplex linear substrates, the enzyme has a strict 5'----3' polarity with respect to the single strand to which it binds. Nicked circular DNA is not utilized as a substrate, and the enzyme requires single-stranded gaps between 5 and 21 nucleotides long to unwind oligonucleotide fragments from partially duplex linear molecules. The enzyme also requires duplex regions at least 11 base pairs long when these are present at the ends of linear molecules. Rad3 DNA helicase activity is inhibited by the presence of ultraviolet-induced photoproducts in duplex regions of partially duplex circular molecules.

Adenosine Triphosphatases↗
PubMed1989

Regulation of the RAD2 gene of Saccharomyces cerevisiae.

Regulation of the DNA damage-inducible RAD2 gene was investigated in yeast cells transformed with centromeric plasmids containing RAD2-lacZ fusion constructs. Deletion analysis defined several regions in the 350bp region upstream of the translational start codon which are required for induction of beta-galactosidase activity. No deletions resulted in constitutively enhanced expression. We therefore conclude that induction of RAD2 by DNA-damaging agents is positively regulated. Two domains required for induction have a similar sequence and are located approximately 70 and approximately 140bp upstream of the major transcriptional start site. Four other sequence domains required for induction contain uninterrupted poly(dA) poly(dT) stretches 9-13bp long. Deletion of some of these AT-rich domains also affects constitutive expression of RAD2. Expression of RAD2 is not cell-cycle-regulated in mitotic cells. However, meiosis is accompanied by increased steady-state levels of RAD2 mRNA in the absence of DNA damage. This enhanced transcription is not dependent on the presence of upstream sequences required for regulation of induction by DNA damage. Increased steady-state levels of RAD2 mRNA are induced by cycloheximide in asynchronously dividing populations of cells, but not in non-replicating cells arrested in G1 phase of the cell cycle. Following exposure to u.v. irradiation induction is also dramatically reduced in non-replicating cells.

Base Sequence↗
PubMed1989

Nucleotide sequence of the wild-type RAD4 gene of Saccharomyces cerevisiae and characterization of mutant rad4 alleles.

Shuttle plasmids carrying the wild-type RAD4 gene of Saccharomyces cerevisiae cannot be propagated in Escherichia coli (R. Fleer, W. Siede, and E. C. Friedberg, J. Bacteriol. 169:4884-4892, 1987). In order to determine the nucleotide sequence of the cloned gene, we used a plasmid carrying a mutant allele that allows plasmid propagation in E. coli. The wild-type sequence in the region of this mutation was determined from a second plasmid carrying a different mutant rad4 allele. We established the locations and characteristics of a number of spontaneously generated plasmid-borne RAD4 mutations that alleviate the toxicity of the wild-type gene in E. coli and of several mutagen-induced chromosomal mutations that inactivate the excision repair function of RAD4. These mutations are situated in very close proximity to each other, and all are expected to result in the expression of truncated polypeptides missing the carboxy-terminal one-third of the Rad4 polypeptide. This region of the gene may be important both for the toxic effect of the Rad4 protein in E. coli and for its role in DNA repair in S. cerevisiae.

Alleles↗
PubMed1989

Human chromosome 15 confers partial complementation of phenotypes to xeroderma pigmentosum group F cells.

Microcell-mediated transfer of a single human chromosome from repair-proficient human cells to genetic complementation group F cells from the hereditary disease xeroderma pigmentosum (XP) results in partial complementation of repair-defective phenotypes. The complementing chromosome was identified by cytogenetic and molecular analysis as human chromosome 15. Transfer of this chromosome to XP-F cells restores approximately 20% of the resistance of wild-type cells to killing by UV radiation or by the UV-mimetic chemical 4-nitroquinoline-1-oxide (4NQO), as well as partial repair synthesis of DNA measured as unscheduled DNA synthesis. Additionally, complemented XP-F cells have an enhanced capacity for reactivation of the plasmid-borne E. coli cat gene following its inactivation by UV radiation. Phenotypic complementation of XP cells by chromosome 15 is specific to genetic complementation group F; no effect on the UV sensitivity of XP-A, XP-C, or XP-D cells was detected. The observation that phenotypic complementation is partial is open to several interpretations and does not allow the definitive conclusion that the XP-F locus is carried on chromosome 15.

Cell Division↗
PubMed1988

A yeast DNA repair gene partially complements defective excision repair in mammalian cells.

The RAD10 gene of Saccharomyces cerevisiae is required for nucleotide excision repair of DNA. Expression of RAD10 mRNA and Rad10 protein was demonstrated in Chinese hamster ovary (CHO) cells containing amplified copies of the gene, and RAD10 mRNA was also detected in stable transfectants without gene amplification. Following transfection with the RAD10 gene, three independently isolated excision repair-defective CHO cell lines from the same genetic complementation group (complementation group 2) showed partial complementation of sensitivity to killing by UV radiation and to the DNA cross-linking agent mitomycin C. These results were not observed when RAD10 was introduced into excision repair-defective CHO cell lines from other genetic complementation groups, nor when the yeast RAD3 gene was expressed in cells from genetic complementation group 2. Enhanced UV resistance in cells carrying the RAD10 gene was accompanied by partial reactivation of the plasmid-borne chloramphenicol acetyltransferase (cat) gene following its inactivation by UV radiation. The phenotype of CHO cells from genetic complementation group 2 is also specifically complemented by the human ERCC1 gene, and the ERCC1 and RAD10 genes have similar amino acid sequences. The present experiments therefore indicate that the structural homology between the yeast Rad10 and human Ercc1 polypeptides is reflected at a functional level, and suggest that nucleotide excision repair proteins are conserved in eukaryotes.

Animals↗
PubMed1988

Rad3 protein of Saccharomyces cerevisiae: overexpression and preliminary characterization using specific antibodies.

The cloned RAD3 gene of Saccharomyces cerevisiae was tailored into expression vectors for overexpression of Rad3 protein in Escherichia coli and in yeast. In both organisms the overexpressed protein is detected as a species of molecular weight ca. 90 kDa, the size expected from the sequence of the cloned gene. The protein overexpressed in E. coli is largely insoluble; however the insoluble fraction was used to generate affinity-purified polyclonal antisera which proved to be powerful reagents for the initial characterization of Rad3 protein expressed in yeast. These studies showed that: (1) when overexpressed in yeast most of the Rad3 protein is detected in the soluble fraction of cell extracts; (2) endogenous Rad3 protein is untransformed cells is also ca. 90 kDa in size and is located in the cell nucleus; (3) Rad3/beta-galactosidase fusion protein partially purified on an affinity matrix is associated with DNA-dependent ATPase activity that is inhibited in the presence of anti-Rad3 antibodies, suggesting that Rad3 protein is an ATPase; and (4) Rad3 antibodies cross-react with two electrophoretically distinguishable polypeptides present in the nuclear fraction of human cells, and with a single polypeptide in extracts of Drosophila cells.

Adenosine Triphosphatases↗
PubMed1987

Overexpression of the RAD2 gene of S. cerevisiae: identification and preliminary characterization of Rad2 protein.

The cloned RAD2 gene of S. cerevisiae was tailored into regulatable expression vectors for overexpression of Rad2 protein in E. coli and in yeast. In E. coli both Rad2/beta-galactosidase fusion protein and native Rad2 protein are insoluble, but are extractable with 1% Sarkosyl. In yeast some of the overexpressed native Rad2 protein is also insoluble; however, soluble protein is readily detected by immunoblotting with Rad2-specific antibodies. All forms of the protein detected in transformed or untransformed yeast cells and the insoluble species in E. coli migrate in denaturing polyacrylamide gels with an apparent molecular weight considerably larger than the size predicted from the sequence of the RAD2 coding region. This property is not the result of post-translational glycosylation detectable by binding of concanavalin A, or of phosphorylation of the protein. Overexpression of the RAD2 gene is toxic to yeast. Transformed yeast cells grow much more slowly than untransformed controls and when yeast transformants are serially propagated cultures show considerably colony heterogeneity and concomitant selection for rapidly growing variants which express less Rad2 protein. Antisera raised against Rad2/beta-galactosidase fusion protein expressed in E. coli do not cross-react with Rad1, Rad3 or Rad10 protein in crude extracts of yeast, nor with purified E. coli UvrA, UvrB or UvrC proteins.

Animals↗