Xeroderma pigmentosum, Cockayne's syndrome, helicases, and DNA repair: what's the relationship?
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Animals↗
Biomedical subjects
E C Friedberg
Publications and source records attributed to E C Friedberg.
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↗
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↗
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↗
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↗
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↗
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↗
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↗
Nuclear targeting sequences.
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Amino Acid Sequence↗
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↗
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↗
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↗
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↗
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↗
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↗
Microcell-mediated chromosome transfer: a strategy for studying the genetics and molecular pathology of human hereditary diseases with abnormal responses to DNA damage.
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Animals↗
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↗
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↗