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Results for “Excision Repair”

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PubMed1975

Dependence upon growth medium and the polA, polC, recA, recB, recC, and exrA genes of separate branches of the uvr Gene-dependent excision-repair process in Escherichia coli K12 cells.

The repair of single-strand breaks which arise in DNA during the uvr gene-dependent excision-repair process was examined in certain radiation-sensitive strains of Escherichia coli K12. The results suggest that the excision-repair process may be divided into two branches, one which is dependent on the presence of growth medium and also the rec+ exr+ genotype, and a second which can occur in buffer (growth medium independent) and is largely dependent on DNA polymerase I. DNA polymerase III appears to be involved in the growth-medium-dependent branch of excision repair, and also in the residual growth-medium-independent repair which occurs in polA1 cells. Chloramphenicol, dinitrophenol, and impurities present in some brands of agar all appear to inhibit the growth-medium-dependent branch of excision-repair. The similarities of the two branches of excision-repair to two known pathways for the repair of X-ray-induced DNA chain breaks are discussed.

Chloramphenicol↗

PubMed2001

Nucleotide excision repair gene XPD polymorphisms and genetic predisposition to melanoma.

The nucleotide excision repair pathway has evolved to deal with UV light-induced DNA damage. Individuals with the rare inherited nucleotide excision repair deficiency disease xeroderma pigmentosum have a 1000-fold increased incidence of skin cancer. We are interested in the possibility that more subtle changes in nucleotide excision repair genes, resulting in either a reduced capacity for repair or in altered interactions between repair proteins and components of the cell cycle control machinery, might constitute important genetic risk factors for the development of skin cancer in the general population. To investigate this hypothesis we have compared the frequency of polymorphisms in exons 6, 22 and 23 of the XPD gene in melanoma patients and a control group. For each of these two allele polymorphisms one of the alleles was over-represented in the melanoma group and there was a significant association with melanoma. Importantly, this association did not extend to markers immediately flanking the XPD gene, thus providing evidence that XPD gene polymorphisms might predispose to melanoma in the general population. There is a report that one of the polymorphic XPD alleles (exon 23 Lys), which is over-represented in the melanoma group, has reduced repair proficiency and we discuss the possibility that this is the causal change to the XPD gene that predisposes to melanoma.

Alleles↗

PubMed1995

The other function of DNA photolyase: stimulation of excision repair of chemical damage to DNA.

DNA photolyase is a light-dependent DNA repair enzyme. It binds to cyclobutane pyrimidine dimers in DNA and upon excitation with a blue light photon splits the cyclobutane ring and restores the pyrimidines to native forms. The enzyme is specific for pyrimidine dimers, and it is not known to catalyze any other reaction either in ground or in excited state. However, when photolyase binds to but cannot catalyze repair because of lack of photoreactivating light, it still aids DNA repair by stimulating the nucleotide excision repair system. Recently, it was found that yeast photolyase binds to other lesions in DNA. In particular, the binding to cisplatin damaged DNA was highly specific. However, in vivo experiments revealed that this binding, in contrast to binding, did not stimulate but actually inhibited the removal of cisplatin damage by excision repair and hence photolyase sensitized cells to killing by cisplatin. In the present study, it is demonstrated that Escherichia coli DNA photolyase binds specifically to cisplatin 1,2-d(GpG) intrastrand cross-link and stimulates the removal of the lesion by E. coli excision nuclease in vitro. In agreement with the in vitro data, in vivo experiments revealed that photolyase makes cells more resistant to cisplatin killing.

Binding Sites↗

PubMed2003

Age-related base excision repair activity in mouse brain and liver nuclear extracts.

To assess DNA repair activity relative to age, in vitro base excision repair assays were performed using brain and liver nuclear extracts prepared from mice of various ages. An 85% decline in repair activity was observed in brain nuclear extracts and a 50% decrease in liver nuclear extracts prepared from old mice compared with 6-day-old mice. Brain nuclear extracts prepared from old mice showed a decreased abundance of DNA polymerase-beta, but the addition of purified protein did not restore base excision repair activity. Abundances of other tested base excision repair proteins did not change relative to age. The conclusion is that, during aging, a decline in DNA repair could contribute to increased levels of DNA damage and mutagenesis.

Aging↗

PubMed1982

Alterations in chromatin structure during DNA excision repair.

Work from a number of laboratories recently has demonstrated that alterations in chromatin structure occur during excision repair in mammalian cells. It is now clear that when cells are damaged with a wide variety of chemical agents or ultraviolet radiation, almost all of the repair synthesis is initially sensitive to staphylococcal nuclease. With time, there is a redistribution of the counts incorporated during excision repair synthesis so that many of them become nuclease resistant and associated with nucleosome core length DNA. In our laboratory, we have demonstrated this phenomenon in human cells damaged with N-acetoxy-2-acetylaminofluorene, 7-bromomethylbenz[a]anthracene, and ultraviolet radiation. It is clear from the work of others that the phenomenon is not unique to human cells since African green monkey cells damaged with either ultraviolet radiation or angelicin also show an initial nuclease sensitivity of repair-incorporated nucleotides follow by rearrangement. Two models to explain these observations have been proposed; one suggests that there is an unfolding of nucleosomes during excision repair followed by a refolding, while the other suggests that sliding of core proteins with respect to DNA occurs during excision repair. These models, as well as recent data bearing on them, will be discussed.

Animals↗

PubMed1993

Re-evaluation of excision repair in the mus304, mus306 and mus308 mutants of Drosophila.

The excision repair capacity of the third chromosomal mus mutations of Drosophila has been re-evaluated. A partial deficiency in the excision repair of pyrimidine dimers originally observed in the mus304 mutants is now attributed to the presence of a secondary phr mutation in that stock. Since the mus306 and mus308 stocks also carry secondary phr mutations, their partial deficiency in repair of pyrimidine dimers may also be the result of that secondary mutation. Accordingly, the Drosophila mutations that are now definitively associated with defects in the incision step of pyrimidine dimers removal are mei-9, mus201 and phr. The genes mus302 and mus310 appear to play a role in later stages of excision repair.

Animals↗

PubMed1986

Nucleotide excision repair genes from the yeast Saccharomyces cerevisiae.

The genetics of nucleotide excision repair in the yeast Saccharomyces cerevisiae is complex, apparently requiring at least 10 genes. We have isolated 5 of these genes (designated RAD1, RAD2, RAD3, RAD4, and RAD10) by molecular cloning and plan to overexpress them in order to generate proteins for biochemical study. We have sequenced four of these five genes and have noted regions of homology with other proteins in the predicted amino acid sequence of some of them. In particular, there is striking homology between Rad3 protein and a number of prokaryotic and eukaryotic proteins that bind nucleotides and hydrolyze ATP or GTP. Mutations in this region of the RAD3 gene render cells defective in the nucleotide excision repair function. In addition to its role in nucleotide excision repair, the RAD3 gene is essential for the viability of haploid cells in the absence of DNA damage. The nature of the essential function is unknown. The RAD1 and RAD3 genes are not inducible by DNA damaging agents. However, exposure of cells to UV radiation, 4-nitroquinoline 1-oxide, or gamma radiation results in 4- to 6-fold enhanced expression of the RAD2 gene.

Amino Acid 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↗

PubMed1994

Cloning and molecular characterization of the Chinese hamster ERCC2 nucleotide excision repair gene.

The Chinese hamster ERCC2 nucleotide excision repair gene, encoding a presumed ATP-dependent DNA helicase, was cloned from the V79 cell line, and its nucleotide sequence was determined. The approximately 15-kb gene comprises 23 exons with a 2283-base open reading frame. The predicted 760-amino-acid protein is 98% identical to the human ERCC2/XPD (760 amino acids), 51% identical to the Saccharomyces cerevisiae RAD3 (778 amino acids), and 54% identical to the Schizosaccharomyces pombe rad15 (772 amino acids) proteins. The promoter region of the hamster ERCC2 gene contains a pyrimidine-rich stretch (42 nucleotides, 88% C+T) similar to sequences found in the promoter regions of two other nucleotide excision repair genes, a GC box, a putative alpha-Pal transcription factor binding site, and two CAAT boxes. There is no apparent TAATA box. No consensus polyadenylation sequence (AATAAA or its variants) was found within 663 bases 3' of the translation termination codon.

Adenosine Triphosphatases↗

PubMed2003

p53 and regulation of DNA damage recognition during nucleotide excision repair.

In response to a variety of types of DNA damage, the p53 tumor suppressor gene product is activated and regulates a number of downstream cellular processes such as cell cycle arrest, apoptosis and DNA repair. Recent discoveries concerning the regulation of DNA repair processes by p53, such as nucleotide excision repair (NER) and base excision repair (BER) have paved the way for studies to understand the mechanisms governing p53-dependent DNA repair. Although several theories have been proposed, accumulating evidence points to a transcriptional regulatory role for p53 in NER, mediating expression of the global genomic repair (GGR)-specific damage recognition genes, DDB2 and XPC. In BER, a more direct role for p53 has been proposed, potentially acting through protein-protein interactions with BER specific factors. These advances have greatly enhanced our understanding of the role of p53 in DNA repair and this review comprehensively summarizes current opinions on the mechanisms of p53-dependent DNA repair.

BRCA1 Protein↗

PubMed1996

Influence of the uvr-dependent nucleotide excision repair on DNA adducts formation and mutagenic spectrum of a potent genotoxic agent: 7-methoxy-2-nitronaphtho[2,1-b]furan (R7000).

The influence of the uvr-dependent excision repair system on the lethal action, mutagenic specificity, SOS induction and DNA adducts formation of 7-methoxy-2-nitronaphtho[2,1-b]furan (R7000), a potent genotoxic nitrofuran, were examined in Escherichia coli. Binding measurements of 3H-labelled R7000 to DNA indicated that R7000-DNA adducts can be removed by excision repair soon after the action of the chemical: 50% of the DNA adducts were removed within 10 min of treatment. After 1 h of incubation the level of excision reached 70%. This result was confirmed using the postlabelling technique. We found that R7000 yielded at least 10 different DNA adducts. Each of the adducts detected could be removed by excision repair. The rates of excision appeared different from one to the other. In addition, using a lacZ reversion system that is able to detect each type of base substitution mutations [1], we found that in uvrA bacteria deficient in excision repair, R7000 can induce 5 out of the 6 possible mutational events: GC-->TA, AT-->TA, GC-->CG, AT-->CG and GC-->AT. The transition AT-->GC was not observed. Only 3 transversions: GC-->TA, AT-->TA and GC-->CG could be detected in repair proficient uvr+ bacteria. The differences between the mutagenic spectra obtained in either uvr+ bacteria or uvrA mutants indicate that some potentially mutagenic DNA adducts induced by R7000 can be removed by excision repair, thus lowering the mutagenic potency of the chemical and modifying the mutagenic spectrum detected.

Bacterial Proteins↗

PubMed2006

The role of Schizosaccharomyces pombe DNA repair enzymes Apn1p and Uve1p in the base excision repair of apurinic/apyrimidinic sites.

In Schizosaccharomyces pombe the repair of apurinic/apyrimidinic (AP) sites is mainly initiated by AP lyase activity of DNA glycosylase Nth1p. In contrast, the major AP endonuclease Apn2p functions by removing 3'-alpha,beta-unsaturated aldehyde ends induced by Nth1p, rather than by incising the AP sites. S. pombe possesses other minor AP endonuclease activities derived from Apn1p and Uve1p. In this study, we investigated the function of these two enzymes in base excision repair (BER) for methyl methanesulfonate (MMS) damage using the nth1 and apn2 mutants. Deletion of apn1 or uve1 from nth1Delta cells did not affect sensitivity to MMS. Exogenous expression of Apn1p failed to suppress the MMS sensitivity of nth1Delta cells. Although Apn1p and Uve1p incised the oligonucleotide containing an AP site analogue, these enzymes could not initiate repair of the AP sites in vivo. Despite this, expression of Apn1p partially restored the MMS sensitivity of apn2Delta cells, indicating that the enzyme functions as a 3'-phosphodiesterase to remove 3'-blocked ends. Localization of Apn1p in the nucleus and cytoplasm hints at an additional function of the enzyme other than nuclear DNA repair. Heterologous expression of Saccharomyces cerevisiae homologue of Apn1p completely restored the MMS resistance of the nth1Delta and apn2Delta cells. This result confirms a difference in the major pathway for processing the AP site between S. pombe and S. cerevisiae cells.

Bleomycin↗

PubMed2000

Repair of UV damage in plants by nucleotide excision repair: Arabidopsis UVH1 DNA repair gene is a homolog of Saccharomyces cerevisiae Rad1.

To analyze plant mechanisms for resistance to UV radiation, mutants of Arabidopsis that are hypersensitive to UV radiation (designated uvh and uvr) have been isolated. UVR2 and UVR3 products were previously identified as photolyases that remove UV-induced pyrimidine dimers in the presence of visible light. Plants also remove dimers in the absence of light by an as yet unidentified dark repair mechanism and uvh1 mutants are defective in this mechanism. The UVH1 locus was mapped to chromosome 5 and the position of the UVH1 gene was further delineated by Agrobacterium-mediated transformation of the uvh1-1 mutant with cosmids from this location. Cosmid NC23 complemented the UV hypersensitive phenotype and restored dimer removal in the uvh1-1 mutant. The cosmid encodes a protein similar to the S. cerevisiae RAD1 and human XPF products, components of an endonuclease that excises dimers by nucleotide excision repair (NER). The uvh1-1 mutation creates a G to A transition in intron 5 of this gene, resulting in a new 3' splice site and introducing an in-frame termination codon. These results provide evidence that the Arabidopsis UVH1/AtRAD1 product is a subunit of a repair endonuclease. The previous discovery in Lilium longiflorum of a homolog of human ERCC1 protein that comprises the second subunit of the repair endonuclease provides additional evidence for the existence of the repair endonuclease in plants. The UVH1 gene is strongly expressed in flower tissue and also in other tissues, suggesting that the repair endonuclease is widely utilized for repair of DNA damage in plant tissues.

Amino Acid 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↗

PubMed1985

Inhibition of excision repair of DNA in u.v.-irradiated Escherichia coli by phenethyl alcohol.

Membrane-specific drugs such as procaine and chlorpromazine have been shown to inhibit excision repair of DNA in u.v.-irradiated E. coli. One possible mechanism is that, if association of DNA with the cell membrane is essential for excision repair, this process may be susceptible to drugs affecting the structure of cell membranes. We examined the effect of phenethyl alcohol, which is a membrane-specific drug and known to dissociate the DNA-membrane complex, on excision repair of DNA in u.v.-irradiated E. coli cells. The cells were irradiated with u.v. light and then held at 30 degrees C in buffer (liquid-holding) in the presence or absence of phenethyl alcohol. It was found that phenethyl alcohol inhibits the liquid-holding recovery in both wild-type and recA strains, corresponding to its dissociating action on the DNA-membrane complex. Thus, the association of DNA with cell membrane is an important factor for excision repair in E. coli. Procaine did not show the dissociating effect, suggesting that at least two different mechanisms are responsible for the involvement of cell membrane in excision repair of DNA in E. coli.

Cell Membrane↗

PubMed2001

Excision repair of adozelesin-N3 adenine adduct by 3-methyladenine-DNA glycosylases and UvrABC nuclease.

Adozelesin is a synthetic analog of the antitumor antibiotic CC-1065, which alkylates the N3 of adenine in the minor groove in a sequence-selective manner. Since the cytotoxic potency of a DNA alkylating agent can be modulated by DNA excision repair system, we investigated whether nucleotide excision repair (NER) and base excision repair (BER) enzymes are able to excise the bulky DNA adduct induced by adozelesin. The UvrABC nuclease and 3-methyladenine-DNA glycosylase, that exhibit a broad spectrum of substrate specificity, were selected as typical NER and BER enzymes, respectively. The adozelesin-DNA adduct was first formed in the radiolabeled restriction DNA fragment and its excision by purified repair enzymes was monitored on a DNA sequencing gel. The treatment of the DNA adduct with a purified UvrABC nuclease and sequencing gel analysis of cleaved DNA showed that UvrABC nuclease was able to incise the adozelesin adduct. The incision site corresponded to the general nuclease incision site. Excision of this adduct by 3-methyladenine-DNA glycosylases was determined following the treatment of the DNA adduct with a homogeneous recombinant bacterial, rat and human 3-methyladenine-DNA glycosylases. Abasic sites generated by DNA glycosyalses were cleaved by the associated lyase activity of the E. coli formamidopyrimidine-DNA glycosylase (Fpg). Resolution of cleaved DNA on a sequencing gel showed that the DNA glycosylase from different sources could not release the N3-adenine adducts. A cytotoxicity assay using E. coli repair mutant strains showed that E. coli mutant strains defective in the uvrA gene were more sensitive to cell killing by adozelesin than E. coli mutant strain defective in the alkA gene or the wild type. These results suggest that the NER pathway seems to be the major excision repair system in protecting cells from the cytotoxicity of adozelesin.

Alkylation↗

PubMed1995

RPA involvement in the damage-recognition and incision steps of nucleotide excision repair.

Human replication protein (RPA) functions in DNA replication, homologous recombination and nucleotide excision repair. This multisubunit single-stranded DNA-binding protein may be required to make unique protein-protein contacts because heterologous single-stranded binding proteins cannot substitute for RPA in these diverse DNA transactions. We report here that, by using affinity chromatography and immunoprecipitation, we found that human RPA bound specifically and directly to two excision repair proteins, the xeroderma pigmentosum damage-recognition protein XPA (refs 8, 9) and the endonuclease XPG (refs 10-13). Although it had been suggested that RPA might function before the DNA synthesis repair stage, our finding that a complex of RPA and XPA showed a striking cooperativity in binding to DNA lesions indicates that RPA may function at the very earliest stage of excision repair. In addition, by binding XPG, RPA may target this endonuclease to damaged DNA.

Chromatography, Affinity↗

PubMed2000

Excision of tamoxifen-DNA adducts by the human nucleotide excision repair system.

The antiestrogen tamoxifen is used in the treatment of breast cancer and has recently been recommended as a chemopreventive drug for women at high risk for breast cancer. However, women treated with the drug have an increased incidence of endometrial cancer. It has been suggested that this endometrial cancer might result from mutagenic DNA adducts, which are formed by electrophilic tamoxifen species generated by metabolic activation of the drug. Because the frequency of damage-induced mutations is strongly dependent on the repairability of the lesion, we investigated the repair of the major tamoxifen-DNA adducts by the human nucleotide excision repair system. Using the reconstituted human excision repair system and synthetic DNA substrates, we found that the four types of tamoxifen-DNA adducts detected in the endometrium were repaired with moderate to poor efficiency by nucleotide excision repair. It is concluded that individual variations in repair capacity may play a role in the development of tamoxifen-induced endometrial cancer.

Antineoplastic Agents, Hormonal↗