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Results for “Excision Repair”

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PubMed1991

Mutational specificity of ethyl methanesulfonate in excision-repair-proficient and -deficient strains of Drosophila melanogaster.

The vermilion gene was used as a target to determine the mutational specificity of ethyl methanesulfonate (EMS) in germ cells of Drosophila melanogaster. To study the impact of DNA repair on the type of mutations induced, both excision-repair-proficient (exr+) and excision-repair-deficient (exr-) strains were used for the isolation of mutant flies. In all, 28 mutants from the exr+ strain and 24 from the exr- strain, were characterized by sequence analysis. In two mutants obtained from the exr+ strain, small deletions were observed. All other mutations were caused by single base-pair changes. In two mutants double base-pair substitutions had occurred. Of the mutations induced in the exr+ strain, 22 (76%) were GC----AT transitions, 3 (10%) AT----TA transversions, 2 (6%) GC----TA transversions and 2 (6%) were deletions. As in other systems, the mutation spectrum of EMS in Drosophila is dominated by GC----AT transitions. Of the mutations in an exr- background, 12 (48%) were GC----TA transitions, 7 (28%) AT----TA transversions, 5 (20%) GC----TA transversions and 1 (4%) was a AT----GC transition. The significant increase in the contribution of transversion mutations obtained in the absence of an active maternal excision-repair mechanism, clearly indicates efficient repair of N-alkyl adducts (7-ethyl guanine and 3-ethyl adenine) by the excision-repair system in Drosophila germ cells.

Animals↗

PubMed1999

Role of DNA polymerase beta in the excision step of long patch mammalian base excision repair.

The two base excision repair (BER) subpathways in mammalian cells are characterized by the number of nucleotides synthesized into the excision patch. They are the "single-nucleotide" BER pathway and the "long patch" (several nucleotides incorporated) BER pathway. Both of these subpathways involve excision of a damaged base and/or nearby nucleotides and DNA synthesis to fill the excision gap. Whereas DNA polymerase beta (pol beta) is known to participate in the single-nucleotide BER pathway, the identity of polymerases involved in long patch BER has remained unclear. By analyzing products of long patch excision generated during BER of a uracil-containing DNA substrate in mammalian cell extracts we find that long patch excision depends on pol beta. We show that the excision of the characteristic 5'-deoxyribose phosphate containing oligonucleotide (dRP-oligo) is deficient in extracts from pol beta null cells and is rescued by addition of purified pol beta. Also, pol beta-neutralizing antibody inhibits release of the dRP-oligo in wild-type cell extracts, and the addition of pol beta after inhibition with antibody completely restores the excision reaction. The results indicate that pol beta plays an essential role in long patch BER by conducting strand displacement synthesis and controlling the size of the excised flap.

Animals↗

PubMed1991

Routine autoradiographic analysis of DNA excision-repair. Report of prenatal and postnatal diagnosis in eleven families.

DNA excision-repair of UV induced damages was investigated by unscheduled DNA synthesis and quantitative autoradiography. The method has been routinely used on lymphocytes for postnatal diagnosis of xeroderma pigmentosum and PIBIDS syndrome. Ten XP-families including 13 clinical XP patients and 9 XP-risk children, and one family with one clinical PIBIDS case and one PIBIDS-risk child were screened. Each of the 14 affected patients were biologically ascertained with a significant excision-repair defect. Among the 9 XP-risk children without clinical manifestations, the DNA excision-repair was defected in 4 cases considered as biological XP, and normal in 5 cases considered as biologically normal subjects. Likewise the PIBIDS-risk child exhibited a normal excision-repair. According to the age of the XP or PIBIDS-risk children, and the delay of appearance of clinical manifestations, the method should not present neither false positive nor false negative results and allows the infraclinical diagnosis. The protocol was extended for prenatal diagnosis on amniocytes and fetal cord blood. Excision-repair analysis on normal cultivated chorionic villi cells has been performed allowing a further first trimester prenatal diagnosis.

Amniotic Fluid↗

PubMed2001

Controlling the efficiency of excision repair.

The early studies are recounted, that led to the discovery of the ubiquitous process of DNA excision repair, followed by a review of the pathways of transcription-coupled repair (TCR) and global genomic nucleotide excision repair (GGR). Repair replication of damaged DNA in UV-irradiated bacteria was discovered through the use of 5-bromouracil to density-label newly synthesized DNA. This assay was then used in human cells to validate the phenomenon of unscheduled DNA synthesis as a measure of excision repair and to elucidate the first example of a DNA repair disorder, xeroderma pigmentosum. Features of the TCR pathway (that is defective in Cockayne syndrome (CS)) include the possibility of "gratuitous TCR" at transcription pause sites in undamaged DNA. The GGR pathway is shown to be controlled through the SOS stress response in E. coli and through the activated product of the p53 tumor suppressor gene in human cells. These regulatory systems particularly affect the efficiency of repair of the predominant UV-induced photoproduct, the cyclobutane pyrimidine dimer, as well as that of chemical carcinogen adducts, such as benzo(a)pyrene diol-epoxide. Rodent cells (typically lacking the p53-controlled GGR pathway) and tumor virus infected human cells (in which p53 function is abrogated) are unable to carry out efficient GGR of some lesions. Therefore, caution should be exercised in the interpretation of results from such systems for risk assessment in genetic toxicology. Many problems in excision repair remain to be solved, including the mechanism of scanning the DNA for lesions and the subcellular localization of the repair factories. Also there are persisting questions regarding the multiple options of repair, recombination, and translesion synthesis when replication forks encounter lesions in the template DNA. That is where the field of DNA excision repair began four decades ago with studies on the recovery of DNA synthesis in UV-irradiated bacteria.

Animals↗

PubMed1991

How relevant is the Escherichia coli UvrABC model for excision repair in eukaryotes?

Knowledge about the DNA excision repair system is increasing rapidly. A detailed model for this process in Escherichia coli has emerged in which a lesion in the DNA is first recognized by the UvrA2B helicase complex. Subsequently, UvrC mediates incision on both sites of the DNA injury. Finally, the concerted action of helicase II (UvrD), polymerase and ligase takes care of removal of the damage-containing oligonucleotide, DNA resynthesis and sealing of the residual nick. In the eukaryotes, yeast and mammals a total of 10 excision repair genes have been analysed thus far. However, little is still known about the molecular mechanism of this repair reaction. Amino acid sequence comparison suggests that at least three DNA helicases operate in eukaryotic nucleotide excision. In addition, a striking sequence conservation is noted between human and yeast repair proteins. But no eukaryotic homologs of the UvrABC proteins have been identified. In this Commentary the parallels and differences between the prokaryotic and eukaryotic excision repair pathways are weighed in an attempt to assess the relevance of the E. coli model for the eukaryotic system.

DNA Repair↗

PubMed1995

Nucleotide excision repair DNA synthesis by DNA polymerase epsilon in the presence of PCNA, RFC, and RPA.

In eukaryotes, nucleotide excision repair of DNA is a complex process that requires many polypeptides to perform dual incision and remove a segment of about 30 nucleotides containing the damage, followed by repair DNA synthesis to replace the excised segment. Nucleotide excision repair DNA synthesis is dependent on proliferating cell nuclear antigen (PCNA). To study gap-filling DNA synthesis during DNA nucleotide excision repair, UV-damaged DNA was first incubated with PCNA-depleted human cell extracts to create repair incisions. Purified DNA polymerase delta or epsilon, with DNA ligase, was then used to form the repair patch. DNA polymerase delta could perform repair synthesis and was strictly dependent on the presence of both PCNA and replication factor C, but gave rise to a very low proportion of complete, ligated circles. The presence of replication protein A (which is also required for nucleotide excision repair) did not alter this result, while addition of DNase IV increased the fraction of ligated products. DNA polymerase epsilon, on the other hand, could fill the repair patch in the absence of PCNA and replication factor C, and most of the products were ligated circles. Addition of replication protein A changed the situation dramatically, and synthesis by polymerase epsilon became dependent on both PCNA and replication factor C. A combination of DNA polymerase epsilon, PCNA, replication factor C, replication protein A, and DNA ligase I appears to be well-suited to the task of creating nucleotide excision repair patches.

Bacteriophage T4↗

PubMed1992

Efficient repair of O6-ethylguanine, but not O4-ethylthymine or O2-ethylthymine, is dependent upon O6-alkylguanine-DNA alkyltransferase and nucleotide excision repair activities in human cells.

The formation and persistence of O6-ethylguanine, O4-ethylthymine, and O2-ethylthymine were quantitated in the genomic DNA of human lymphoblasts exposed to 1.0 mM N-ethyl-N-nitrosourea using immunoslot-blot. The three cell lines used included one which lacks O6-alkylguanine-DNA alkyltransferase, one deficient in nucleotide excision repair, and a third which is competent in both of these repair pathways. The activity of O6-alkylguanine-DNA alkyltransferase was further modulated with O6-benzylguanine, a specific inhibitor of this protein. Repair of the O-ethylated thymines was slow and not related to either DNA repair phenotype. O6-Ethylguanine was repaired with a half-life of about 8 h in cells which expressed both O6-alkylguanine-DNA alkyltransferase and nucleotide excision repair functions. Cells expressing O6-alkylguanine-DNA alkyltransferase activity but lacking nucleotide excision repair showed only slow repair of O6-ethylguanine (half-life of O6-ethylguanine, 43 h), while cells lacking the alkyltransferase showed little or no repair of O6-ethylguanine regardless of nucleotide excision repair activity (half-lives of O6-ethylguanine, 53 to greater than 100 h). We conclude that O6-alkylguanine-DNA alkyltransferase and nucleotide excision repair cooperate in the repair of O6-ethylguanine in human cells.

B-Lymphocytes↗

PubMed1997

Repair of DNA loops involves DNA-mismatch and nucleotide-excision repair proteins.

A number of enzymes recognize and repair DNA lesions. The DNA-mismatch repair system corrects base-base mismatches and small loops, whereas the nucleotide-excision repair system removes pyrimidine dimers and other helix-distorting lesions. DNA molecules with mismatches or loops can arise as a consequence of heteroduplex formation during meiotic recombination. In the yeast Saccharomyces cerevisiae, repair of mismatches results in gene conversion or restoration, and failure to repair the mismatch results in post-meiotic segregation (PMS). The ratio of gene-conversion to PMS events reflects the efficiency of DNA repair. By examining the PMS patterns in yeast strains heterozygous for a mutant allele with a 26-base-pair insertion, we find that the repair of 26-base loops involves Msh2 (a DNA-mismatch repair protein) and Rad1 (a protein required for nucleotide-excision repair).

Alcohol Oxidoreductases↗

PubMed1988

Transfection of the cloned human excision repair gene ERCC-1 to UV-sensitive CHO mutants only corrects the repair defect in complementation group-2 mutants.

The human DNA-excision repair gene ERCC-1 is cloned by its ability to correct the excision-repair defect of the ultraviolet light- and mitomycin-C-sensitive CHO mutant cell line 43-3B. This mutant is assigned to complementation group 2 of the excision-repair-deficient CHO mutants. In order to establish whether the correction by ERCC-1 is confined to CHO mutants of one complementation group, the cloned repair gene, present on cosmid 43-34, was transfected to representative cell lines of the 6 complementation groups that have been identified to date. Following transfection, mycophenolic acid was used to select for transferants expressing the dominant marker gene Ecogpt, also present on cosmid 43-34. Cotransfer of the ERCC-1 gene was shown by Southern blot analysis of DNA from pooled (500-2000 independent colonies) transformants of each mutant. UV survival and UV-induced UDS showed that only mutants belonging to complementation group 2 and no mutants of other groups were corrected by the ERCC-1 gene. This demonstrates that ERCC-1 does not provide an aspecific bypass of excision-repair defects in CHO mutants and supports the assumption that the complementation analysis is based on mutations in different repair genes.

Animals↗

PubMed1999

The initiation of DNA base excision repair of dipyrimidine photoproducts.

One of the major DNA repair pathways is base excision repair, in which DNA bases that have been damaged by endogenous or exogenous agents are removed by the action of a class of enzymes known as DNA glycosylases. One subset of the known DNA glycosylases has an associated abasic lyase activity that generates a phosphodiester bond scission. The base excision pathway is completed by the sequential action of abasic endonucleases, DNA polymerases, and DNA ligases. Base excision repair of ultraviolet (UV) light-induced dipyrimidine photoproducts has been described in a variety of prokaryotic and eukaryotic organisms and phages. These enzymes vary significantly in their exact substrate specificity and in the catalytic mechanism by which repair is initiated. The prototype enzyme within this class of UV-specific DNA glycosylases is T4 endonuclease V. Endonuclease V holds the distinction of being the first glycosylase (1) to have its structure solved by X-ray diffraction of the enzyme alone as well as in complex with pyrimidine dimer-containing DNA, (2) to have its key catalytic active site residues identified, and (3) to have its mechanism of target DNA site location determined and the biological relevance of this process established. Thus, the study of endonuclease V has been critical in gaining a better understanding of the mechanisms of all DNA glycosylases.

DNA Repair↗

PubMed2004

Expression of the oxidative base excision repair enzymes is not induced in TK6 human lymphoblastoid cells after low doses of ionizing radiation.

Most of the DNA damage produced by ionizing radiation is repaired by the base excision repair (BER) pathway. To determine whether the BER genes were up-regulated by low doses of ionizing radiation, we investigated their expression in TK6 human lymphoblastoid cells by measuring mRNA levels using real-time quantitative PCR. No induction at the transcriptional level of any of the base excision repair genes, NTH1 (NTHL1), OGG1, NEIL1, NEIL2, NEIL3, APE1, POLB, or accessory protein genes, LIG3, XRCC1 or XPG, was found at gamma-radiation doses ranging from 1 cGy to 2 Gy in a 24-h period. As has been measured in other cell lines, a dose-dependent induction of CDKN1A (WAF1) mRNA levels was observed in TK6 cells in the dose range of 0.5 to 2.0 Gy. We also examined BER enzyme activity on 8-oxoguanine-, dihydrouracil- and furan-containing oligonucleotide substrates and found no increase in extracts of TK6 cells after gamma-ray doses of 0.5-2.0 Gy. These data were corroborated by Western blot analysis of APE1 and NTH1, suggesting that the BER enzymes are also not up-regulated at the post-transcriptional level after ionizing radiation exposure.

Blotting, Western↗

PubMed1995

Human DNA repair excision nuclease. Analysis of the roles of the subunits involved in dual incisions by using anti-XPG and anti-ERCC1 antibodies.

Human DNA repair excision nuclease removes DNA damage by incising on both sides of the lesion in a precise manner. The activity requires participation of 16-17 polypeptides. Of these, the XPF.ERCC1 complex and XPG were predicted to carry the nuclease active sites based on studies with the recombinant proteins and the yeast homologs of these proteins. Furthermore, recent work with model (undamaged) substrates have led to predictions of the roles of these proteins in incising 5' or 3' to the lesion. We have used damaged DNA substrates and antibodies to XPG and ERCC1 to test these predictions. Our results reveal that anti-XPG antibodies change the site of 3' incision and at high concentration inhibit the 3' incision without significantly affecting the 5' incision, indicating that XPG makes the 3' incision and further that under this condition 5' incision can occur without 3' incision. In contrast, anti-ERCC1 antibodies inhibit both the 3' and 5' incisions. Using a defined system for excision repair we also demonstrate that the 3' incision can occur without the 5' incision, leading us to conclude that under certain conditions the two incisions can occur independently.

Animals↗

PubMed2004

Therapeutic impact of methoxyamine: blocking repair of abasic sites in the base excision repair pathway.

A major obstacle to effective treatment with alkylating agents in cancer is the presence of elaborate mechanisms of DNA repair. For instance, the methylating therapeutic agent, temozolomide forms O6-methylguanine (O6mG), 7-methylguanine (N7mG) and 3-methyladenine (N3mA) DNA adducts that are repaired by at least two mechanisms. The O6mG DNA adduct, a cytotoxic and genotoxic lesion, is repaired by O6-methylguanine DNA-methyltransferase (MGMT). Thus, MGMT is a major mechanism of resistance to methylating agents. Meanwhile, cell death caused by O6mG adducts is promoted by mismatch repair (MMR) system, such that deficiency in MMR is associated with pronounced resistance to methylating agents. N7mG, the dominant lesions formed by methylating therapeutic agents, and N3mA DNA adducts are removed by the base excision repair (BER) pathway. Efficient BER repair minimizes the impact of these lesions in normal and tumor cells. Thus, only when BER is disrupted, do these abundant N-methylated DNA adducts become highly cytotoxic. Most importantly, BER disruption is able to bypass other resistance factors such as MMR defects and high MGMT activity. BER, in fact, is an important drug-resistant factor because of its variety of substrates and the ability to rapidly and efficiently repair these DNA lesions. However, the impact of both active BER and inactive BER on anticancer therapy has yet to be fully studied. This review will establish the premise that targeting abasic sites formed during BER will disrupt this pathway and enhance the therapeutic efficacy of alkylating agents.

Animals↗

PubMed2003

Assessment of nucleotide excision repair XPD polymorphisms in the peripheral blood of gemcitabine/cisplatin-treated advanced non-small-cell lung cancer patients.

Only about one third of non-small-cell lung cancer (NSCLC) patients respond to cisplatin-based chemotherapy. Cisplatin DNA adducts are commonly repaired through the nucleotide excision repair pathway. The study of rare inherited disorders such as xeroderma pigmentosum and Cockayne syndrome has disclosed that XP genes, including XPD, play an essential role in DNA repair, both in the global genomic repair and in the transcription-coupled repair pathways. XPD polymorphism and decreased expression of XP genes have both been linked to lower DNA repair capacity. ERCC1 overexpression has been associated with cisplatin resistance, and experimental evidence shows a close association between ERCC1 and XPD. In the present study, we have examined XPD polymorphisms at codons 751 and 312 in DNA isolated from peripheral blood in 39 patients with gemcitabine/cisplatin-treated locally advanced non-small-cell lung cancer Although no significant correlation was observed between XPD genotype and objective response, a trend toward better response was observed in patients with XPD polymorphism at codon 312. The map of the nucleotide excision repair pathway can be used to design translational research studies to identify and validate predictive markers of response to cisplatin, and the Spanish Lung Cancer Group has recently accrued 250 gemcitabine/cisplatin-treated NSCLC patients for a prospective assessment of XPD genotype

Journal Article↗

PubMed2006

Nucleotide excision repair and cancer.

Nucleotide excision repair (NER) is the most versatile and best studied DNA repair system in humans. NER can repair a variety of bulky DNA damages including UV-light induced DNA photoproducts. NER consists of a multistep process in which the DNA lesion is recognized and demarcated by DNA unwinding. Then, an approximately 28 bp DNA damage containing oligonucleotide is excised followed by gap filling using the undamaged DNA strand as a template. The consequences of defective NER are demonstrated by three rare autosomal-recessive NER-defective syndromes: xeroderma pigmentosum (XP), Cockayne syndrome (CS), and trichothiodystrophy (TTD). XP patients show severe sun sensitivity, freckling in sun exposed skin, and develop skin cancers already during childhood. CS patients exhibit sun sensitivity, severe neurologic abnormalities, and cachectic dwarfism. Clinical symptoms of TTD patients include sun sensitivity, freckling in sun exposed skin areas, and brittle sulfur-deficient hair. In contrast to XP patients, CS and TTD patients are not skin cancer prone. Studying these syndromes can increase the knowledge of skin cancer development including cutaneous melanoma as well as basal and squamous cell carcinoma in general that may lead to new preventional and therapeutic anticancer strategies in the normal population.

Cockayne Syndrome↗

PubMed2004

Trypanosoma cruzi contains a single detectable uracil-DNA glycosylase and repairs uracil exclusively via short patch base excision repair.

Enzymes involved in genomic maintenance of human parasites are attractive targets for parasite-specific drugs. The parasitic protozoan Trypanosoma cruzi contains at least two enzymes involved in the protection against potentially mutagenic uracil, a deoxyuridine triphosphate nucleotidohydrolase (dUTPase) and a uracil-DNA glycosylase belonging to the highly conserved UNG-family. Uracil-DNA glycosylase activities excise uracil from DNA and initiate a multistep base-excision repair (BER) pathway to restore the correct nucleotide sequence. Here we report the biochemical characterisation of T.cruzi UNG (TcUNG) and its contribution to the total uracil repair activity in T.cruzi. TcUNG is shown to be the major uracil-DNA glycosylase in T.cruzi. The purified recombinant TcUNG exhibits substrate preference for removal of uracil in the order ssU>U:G>U:A, and has no associated thymine-DNA glycosylase activity. T.cruzi apparently repairs U:G DNA substrate exclusively via short-patch BER, but the DNA polymerase involved surprisingly displays a vertebrate POLdelta-like pattern of inhibition. Back-up UDG activities such as SMUG, TDG and MBD4 were not found, underlying the importance of the TcUNG enzyme in protection against uracil in DNA and as a potential target for drug therapy.

Amino Acid Sequence↗

PubMed1998

Nucleotide excision repair in the third kingdom.

Nucleotide excision repair, a general repair mechanism for removing DNA damage, is initiated by dual incisions bracketing the lesion. In procaryotes, the dual incisions result in excision of the damage in 12- to 13-nucleotide-long oligomers, and in eucaryotes they result in excision of the damage in the form of 24- to 32-nucleotide-long oligomers. We wished to find out if Archaea perform excision repair. Using cell extracts from Methanobacterium thermoautotrophicum, we found that this organism removes UV-induced (6-4) photoproducts in the form of 10- to 11-mers by incising the sixth to seventh phosphodiester bond 5' to the damage and the fourth phosphodiester bond 3' to the damage.

Animals↗

PubMed1986

Analysis of the essential and excision repair functions of the RAD3 gene of Saccharomyces cerevisiae by mutagenesis.

The RAD3 gene of Saccharomyces cerevisiae, which is involved in excision repair of DNA and is essential for cell viability, was mutagenized by site-specific and random mutagenesis. Site-specific mutagenesis was targeted to two regions near the 5' and 3' ends of the coding region, selected on the basis of amino acid sequence homology with known nucleotide binding and with known specific DNA-binding proteins, respectively. Two mutations in the putative nucleotide-binding region and one in the putative DNA-binding region inactivate the excision repair function of the gene, but not the essential function. A gene encoding two tandem mutations in the putative DNA-binding region is defective in both excision repair and essential functions of RAD3. Seven plasmids were isolated following random mutagenesis with hydroxylamine. Mutations in six of these plasmids were identified by gap repair of mutant plasmids from the chromosome of strains with previously mapped rad3 mutations, followed by DNA sequencing. Three of these contain missense mutations which inactivate only the excision repair function. The other three carry nonsense mutations which inactivate both the excision repair and essential functions. Collectively our results indicate that the RAD3 excision repair function is more sensitive to inactivation than is the essential function. Overexpression of wild-type Rad3 protein and a number of rad3 mutant proteins did not affect the UV resistance of wild-type yeast cells. However, overexpression of Rad3-2 protein rendered wild-type cells partially UV sensitive, indicating that excess Rad3-2 protein is dominant to the wild-type form. These and other results suggest that Rad3-2 protein retains its affinity for damaged DNA or other substrates, but is not catalytically active in excision repair.

Amino Acid Sequence↗