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PubMedDate not supplied

Effect of S. cerevisiae APN1 protein on mammalian DNA base excision repair.

Mammalian cells transfected with the S. cerevisiae APN1 protein acquire resistance to oxidizing agents, the damage of which is mainly repaired via DNA base excision repair (BER). We have recently hypothesized that this effect might be linked to the possible capacity of APN1 to accelerate mammalian BER by its 3' diesterase activity. We have investigated here the effect of pure APN1 protein on BER performed by mouse embryonic fibroblast extracts. No significant acceleration was observed in the repair of either a single AP site cleaved by the bifunctional glycosylase NTH of E. coli or the repair of a single 8-oxoguanine, initiated by the bifunctional glycosylase OGG1. Similarly, no significant effect was observed on the repair of a single U (initiated by the monofunctional glycosylase U DNA glycosylase) or the repair of a single natural abasic site. The inability of APN1 to increase the efficiency of BER initiated by bifunctional glycosylases indicates that removal of 3' blocking fragments is not the rate-limiting step of this repair pathway.

Animals↗

PubMed1990

Molecular genetics of eukaryotic DNA excision repair.

DNA repair plays a key role in the prevention of carcinogenesis and mutagenesis. Defective DNA repair has been implicated in various human hereditary disorders that predispose affected individuals to cancer. This article reviews our current understanding of one of major DNA repair systems--the nucleotide excision repair pathway--with special emphasis on the novel findings that have emerged from molecular genetic analysis of yeast and cultured mammalian cells.

Animals↗

PubMedDate not supplied

Excision repair of mouse and human fibroblast cells, and a factor affecting the amount of UV-induced unscheduled DNA synthesis.

Excision-repair ability and the amount of unscheduled DNA synthesis (UDS) after UV irradiation of fibroblast cells (in vitro passage 5) from C57BL mouse embryos were compared with those of human skin fibroblast cells. UDS in the mouse cells was approximately 75% of that in the human cells, although the disappearance of T4 endonuclease-V-susceptible sites and the accumulation of single-strand breaks in the mouse cell DNA indicated that the excision-repair capacity of the mouse cells was 20-35% of that in the human cells. This apparent discrepancy was ascribed to the difference in intracellular dTTP pool size, which was approximately twice as large in the human cells as in the mouse cells. UDS may not be suitable as a quantitative measure of excision repair when comparing the cells from different species.

Animals↗

PubMed1983

Evidence that DNA excision-repair in xeroderma pigmentosum group A is limited but biologically significant.

The loss of pyrimidine dimers in nondividing populations of an excision-repair deficient xeroderma pigmentosum group A strain (XP12BE) was measured throughout long periods (up to 5 months) following exposure to low doses of ultraviolet light (UV, 254 nm) using a UV endonuclease-alkaline sedimentation assay. Excision of about 90% of the dimers induced by 1 J/m2 occurred during the first 50 days. The rate curve has some similarities with that of normal excision-repair proficient cultures that may not be coincidental. Rate curves for both XP12BE and normal cultures are characterized by a fast and slow component, with both rate constants for the XP12BE cultures (0.15 day-1 and 0.025 day-1) a factor of 10 smaller than those observed for the respective components of normal cell cultures. The slow components for both XP12BE and normal cultures extrapolate to about 30% of the initial number of dimers. No further excision was detected throughout an additional 90-day period even though the cultures were capable of excision-repair of other newly-introduced pyrimidine dimers. We conclude that nondividing XP12BE cells in addition to having a slower repair rate, cannot repair some of the UV-induced DNA damage. The repair in XP12BE is shown to have biological significance as detected by a cell-survival assay and dose-fractionation techniques. Nondividing XP12BE cells are more resistant to UV when irradiated chronically than when irradiated acutely with the same total dose.

Cell Survival↗

PubMed1988

Low levels of DNA excision repair in undamaged bovine lymphocytes.

Quiescent and concanavalin A-stimulated bovine lymphocytes were subjected to a buoyant density analysis used in excision repair studies. Despite neutral and alkaline rebands to remove replicative contamination, the CsCl gradient profiles of DNA isolated from unstimulated lymphocytes given a 6-h labelling period revealed a small amount of radioactivity in the normal-density region which is indicative of an excision repair process. It amounted to the incorporation of 8,000-20,000 molecules of thymidine per lymphocyte. In a 12-h labelling period the extent of repair incorporation was twice that measured in a 6-h period. The extent of this repair incorporation was not altered significantly during the initial 6 or 12 h of lectin stimulation when DNA-strand breaks normally present in the unstimulated cells are repaired. The same amount of repair activity was found whether the measurements were made on the same day that the lymphocytes were isolated or on the next day following an overnight incubation of the cells in culture medium. These observations indicate that lymphocytes display a spontaneous excision repair activity that proceeds continuously and at a constant rate.

Animals↗

PubMed2001

In vivo repair of ENU-induced oxygen alkylation damage by the nucleotide excision repair mechanism in Drosophila melanogaster.

DNA damage caused by oxygen alkylation of bases (mainly at O6-G, O4-T and O2-T positions in DNA) has been correlated with the mutagenic and carcinogenic potency of monofunctional alkylating agents. In all kinds of organisms, repair of O6-alkylG is carried out mainly by the enzyme O6-methyl guanine-DNA methyltransferase (MGMT). However, little is known about the repair of the O-alkylT adducts or about the contribution of nucleotide excision repair (NER) to this process, especially in higher eukaryotes. To study the influence of the NER system on the repair of O-alkylation damage, the molecular mutation spectrum induced by N-ethyl-N-nitrosourea (ENU) in an NER-deficient Drosophila strain, carrying a mutation at the mus201 locus, was obtained and compared with a previously published spectrum for NER-proficient conditions. This comparison reveals a clear increase in the frequency of base pair changes, including GC --> AT and AT --> GC transitions and AT --> TA transversions. In addition, one deletion and two frameshift mutations, not found under NER-proficient conditions, were isolated in the NER-deficient mutant. The results demonstrate that: (1) N-alkylation damage contributes considerably (more than 20%) to the mutagenic activity of ENU under NER-deficient conditions, confirming that the NER system repairs this kind of damage; and (2) that in germ cells of Drosophila in vivo, NER seems to repair O6-ethylguanine and/or O2-ethylcytosine, O4-ethylthymine, and possibly also O2-ethylthymine.

Alkylating Agents↗

PubMed1995

An interaction between the DNA repair factor XPA and replication protein A appears essential for nucleotide excision repair.

Replication protein A (RPA) is required for simian virus 40-directed DNA replication in vitro and for nucleotide excision repair (NER). Here we report that RPA and the human repair protein XPA specifically interact both in vitro and in vivo. Mapping of the RPA-interactive domains in XPA revealed that both of the largest subunits of RPA, RPA-70 and RPA-34, interact with XPA at distinct sites. A domain involved in mediating the interaction with RPA-70 was located between XPA residues 153 and 176. Deletion of highly conserved motifs within this region identified two mutants that were deficient in binding RPA in vitro and highly defective in NER both in vitro and in vivo. A second domain mediating the interaction with RPA-34 was identified within the first 58 residues in XPA. Deletion of this region, however, only moderately affects the complementing activity of XPA in vivo. Finally, the XPA-RPA complex is shown to have a greater affinity for damaged DNA than XPA alone. Taken together, these results indicate that the interaction between XPA and RPA is required for NER but that only the interaction with RPA-70 is essential.

Amino Acid Sequence↗

PubMed1991

Preferential excision repair and non-preferential photoreactivation of pyrimidine dimers in the c-ras sequence of cultured goldfish cells.

The time courses of excision repair and photoreactivation of pyrimidine dimers induced by 254-nm UV were examined in the genome overall and in the c-ras sequence of RBCF-1 cells derived from a goldfish, by the use of UV endonuclease of Micrococcus luteus and alkaline agarose gel electrophoresis. Excision repair was more efficient in the ras sequence than in the genome overall, whereas no differences in efficiency of photoreactivation were detected. These results suggest that excision repair is affected by the accessibility of chromatin, while photoreactivation is not.

Animals↗

PubMed1999

The type of DNA glycosylase determines the base excision repair pathway in mammalian cells.

The base excision repair (BER) of modified nucleotides is initiated by damage-specific DNA glycosylases. The repair of the resulting apurinic/apyrimidinic site involves the replacement of either a single nucleotide (short patch BER) or of several nucleotides (long patch BER). The mechanism that controls the selection of either BER pathway is unknown. We tested the hypothesis that the type of base damage present on DNA, by determining the specific DNA glycosylase in charge of its excision, drives the repair of the resulting abasic site intermediate to either BER branch. In mammalian cells hypoxanthine (HX) and 1,N6-ethenoadenine (epsilonA) are both substrates for the monofunctional 3-methyladenine DNA glycosylase, the ANPG protein, whereas 7,8-dihydro-8-oxoguanine (8-oxoG) is removed by the bifunctional DNA glycosylase/beta-lyase 8-oxoG-DNA gly- cosylase (OGG1). Circular plasmid molecules containing a single HX, epsilonA, or 8-oxoG were constructed. In vitro repair assays with HeLa cell extracts revealed that HX and epsilonA are repaired via both short and long patch BER, whereas 8-oxoG is repaired mainly via the short patch pathway. The preferential repair of 8-oxoG by short patch BER was confirmed by the low efficiency of repair of this lesion by DNA polymerase beta-deficient mouse cells as compared with their wild-type counterpart. These data fit into a model where the intrinsic properties of the DNA glycosylase that recognizes the lesion selects the branch of BER that will restore the intact DNA template.

Adenine↗

PubMed1984

Human lymphocytes treated with r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene require low-density lipoproteins for DNA excision repair.

Human lymphocytes which were non-mitogen-stimulated, and which were depleted of lipoproteins, were found to be deficient in DNA excision repair typically initiated in these cells in response to treatment with a direct-acting polynuclear aromatic hydrocarbon carcinogen. Lymphocytes either depleted of lipoproteins or supplemented with human low-density lipoproteins formed DNA-carcinogen adducts which were not chromatographically distinguishable. The state of lipoprotein depletion did not alter lymphocyte uptake of thymidine from the medium. Lymphocytes which were depleted of lipoproteins, treated with carcinogen, and subsequently supplemented with low-density lipoproteins, regained the ability to engage in DNA excision repair. The data suggest that either low-density lipoprotein(s), or a component(s) of low-density lipoprotein(s), is required by human lymphocytes in order to initiate excision repair of carcinogen-damaged DNA.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed1975

Excision-repair of 4-nitroquinolin-1-oxide damage responsible for killing, mutation, and cancer.

Excision-repair of DNA base damage produced by 4-nitroquinoline-1-oxide (4NQO) was compared in Escherichia coli, human cells, and mouse cells. Paper chromotography of acid hydrolysates of DNA extracted from cells treated with 3H-labeled 4NQO revealed four peaks; two kinds of 4NQO-guanine adduct, one kind of 4NQO-adenine adduct, and free 4-aminoquinoline-1-oxide (4AQO). About 80% of the initially formed 4NQO-purine adducts were excised from DNA in E. coli uvrA+ cells during 60 min postincubation, but not at all in uvrA- (excisionless for uv damage) cells. Normal human cells excised about 60% of 4NQO-purine adducts during 24 hr postincubation, but xeroderma pigmentosum (excisionless) cells did not. A mouse cell line susceptible to repair of 4NQO-induced pretransformational damage also showed excision-repair ability for the 4NQO adducts. From these and other results, we conclude that the 4NQO-purine adducts and unstable 4NQO-guanine products (which release 4AQO) are, like pyrimidine dimers, repairable by excision-repair universal among E. coli, mouse, and human being, and that unexcised ones are probably the major cause of killing, mutation, and cancer.

4-Nitroquinoline-1-oxide↗

PubMed1987

Rearrangement of nucleosome structure during excision repair in xeroderma pigmentosum (group A) human fibroblasts.

Rearrangements of chromatin structure during excision repair were examined in xeroderma pigmentosum (XP; complementation group A) human fibroblasts treated with the small-molecule alkylating agent methyl methanesulfonate (MMS). In agreement with past reports, we observed normal levels of repair synthesis in these cells during the first 12 h after exposure to 1.5 mM MMS, in contrast to the near zero incorporation of repair patches following exposure to 12 J/m2 u.v. light. Our results indicate that the relative nuclease sensitivity of newly repaired regions in MMS-treated nuclease sensitivity of newly repaired regions in MMS-treated XP (group A) cells is quantitatively similar to that of newly repaired regions in MMS-treated normal human fibroblasts. This enhanced sensitivity is accompanied by a marked under-representation of repair-incorporated nucleotides in isolated nucleosome core DNA. Pulse-chase experiments demonstrated that these regions rapidly undergo rearrangements in chromatin structure, and both the rate and extent of these rearrangements are similar (but not identical) to those observed in normal cells. This was also the case for the rate and extent of ligation of repair patches, as measured by the sensitivity of these regions to exonuclease III digestion. If the changes in nuclease sensitivity of newly repaired regions in DNA reflect an unfolding of nucleosome structure during excision repair, then these results indicate that the activity associated with this unfolding is present in XP (group A) cells.

DNA↗

PubMed1995

In vitro eukaryotic DNA excision repair assays: an overview.

Great progress is being made in understanding the process of nucleotide excision repair (NER) in eukaryotes. Different lines of research have been developed, among them an in vitro assay with cell-free extracts has played a major role. This in vitro repair assay takes advantage of a cell-free system that can mediate DNA excision-repair by transcriptionally active protein extracts from mammalian cells incubated in the presence of two plasmids of different sizes, one damaged and the other undamaged as internal control. The extent of repair activity is determined by following the level of radiolabeled incorporation during the repair synthesis step consecutive to the excision of DNA lesions. We discuss the interest and drawbacks of this biochemical assay in light of the main results obtained. We report the modifications that we have undertaken in order to determine repair synthesis activity in a chemiluminescent-directed reaction as well as to assess incision activity in protein extracts.

Animals↗

PubMed1995

Phosphorylated and unphosphorylated forms of human single-stranded DNA-binding protein are equally active in simian virus 40 DNA replication and in nucleotide excision repair.

The trimeric human single-stranded DNA-binding protein (HSSB; also called RP-A) plays an essential role in DNA replication, nucleotide excision repair, and homologous DNA recombination. The p34 subunit of HSSB is phosphorylated at the G1/S boundary of the cell cycle or upon exposure of cells to DNA damage-inducing agents including ionizing and UV radiation. We have previously shown that the phosphorylation of p34 is catalyzed by both cyclin-dependent kinase-cyclin A complex and DNA-dependent protein kinase. In this study, we investigated the effect of phosphorylation of p34 by these kinases on the replication and repair function of HSSB. We observed no significant difference with the unphosphorylated and phosphorylated forms of HSSB in the simian virus 40 DNA replication or nucleotide excision repair systems reconstituted with purified proteins. The phosphorylation status of the p34 subunit of HSSB was unchanged during the reactions. We suggest that the phosphorylated HSSB has no direct effect on the basic mechanism of DNA replication and nucleotide excision repair reactions in vitro, although we cannot exclude a role of p34 phosphorylation in modulating HSSB function in vivo through a yet poorly understood control pathway in the cellular response to DNA damage and replication.

DNA Repair↗

PubMed2005

[Polymorphisms in nucleotide excision repair genes XPC and XPD and clinical responses to platinum-based chemotherapy in advanced non-small cell lung cancer].

OBJECTIVE: Nucleotide excision repair has been shown to have great impact on the sensitivity of tumors to platinum-based chemotherapy. This study was to examine the association between genetic polymorphisms in XPC and XPD, two important components in nucleotide excision repair system, and clinical response to platinum-based chemotherapy in advanced non-small cell lung cancer. METHODS: Patients (n = 151) treated with platinum-based chemotherapy were genotyped for the AT dinucleotide insertion or deletion in intron 9 of XPC or Lys751Gln polymorphism in XPD. Clinical response to chemotherapy was obtained after 2 to 3 cycles. The adjusted odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using unconditional logistic regression model and adjusted for sex, age, clinical stage, and regime of chemotherapy. RESULTS: The overall response rate to the chemotherapy (CR + PR) was 35.1%, with 1 CR, 52 PR, 75 SD, and 23 PD. It was found that patients with the XPC LL genotype had significantly higher response rate than patients with the XPC SS genotype (adjusted OR = 3.19, 95% CI = 1.11 - 9.17; P = 0.031). However, no association was found between the XPD Lys751Gln polymorphism and response to the chemotherapy. In addition, these two polymorphisms seemed to have synergic effect, with the OR being 2.90 (95% CI = 1.07 - 7.86) for patients carrying the XPC LL and XPD Lys/Lys genotypes compared with those carrying the XPC SS or SL and XPD Lys/Lys genotypes. CONCLUSION: These results suggest that genetic polymorphisms in nucleotide excision repair might be associated with clinical response to platinum-based chemotherapy.

Adult↗

PubMed2006

Genetic polymorphisms of the XPG and XPD nucleotide excision repair genes in sarcoma patients.

There are more than 50 subtypes of soft tissue sarcomas, among which 30% are associated with specific genetic alterations, including translocations. Several studies have reported associations between cancer risk and polymorphisms of DNA repair genes from the nucleotide excision repair (NER) pathway. NER involves more than 20 proteins whose inactivation leads to xeroderma pigmentosum (XP) or cockayne syndrome (CS), among which XPD, a helicase allowing DNA strand excision by the endonuclease XPG. DNA from 93 patients with synovial sarcomas, myxoid liposarcomas, dermatofibrosarcomas protuberans (DFSP), malignant fibrous histiocytomas and leiomyosarcomas were genotyped for both XPD Lys751Gln and XPG Asp1104His polymorphisms. Departure from Hardy-Weinberg was highly significant for the XPG polymorphism with an excess of heterozygotes in synovial sarcomas (p = 1.5 x 10(-5)), myxoid liposarcomas (p = 1.5 x 10(-4)) and to a lesser extent in DFSP (p = 0.028). In the case of XPD, a significant deviation was observed in synovial sarcomas (p = 3 x 10(-6)) and DFSP (p = 0.0014). When tumors were pooled according to their genetic alterations, the proportion of carriers of the variant XPG allele was significantly increased in sarcomas with specific translocations as compared to sarcomas with complex genetics (p < 10(-9)). No difference was found for XPD. Genotyping of the tumor samples in synovial sarcomas and myxoid liposarcomas revealed frequent loss of heterozygosity for XPG, mostly due to the loss of the frequent allele. For XPD, both alleles were lost with a similar frequency. Our results raise the potential implication of the XPG Asp1104His polymorphism in the occurrence of chromosomal translocations associated with specific subtypes of sarcomas.

Adolescent↗

PubMed1990

Specificity of the mutator effect caused by disruption of the RAD1 excision repair gene of Saccharomyces cerevisiae.

Disruption of RAD1, a gene controlling excision repair in the yeast Saccharomyces cerevisiae, increased the frequency of spontaneous forward mutation in a plasmid-borne copy of the SUP4-o gene. To characterize this effect in detail, a collection of 249 SUP4-o mutations arising spontaneously in the rad1 strain was analyzed by DNA sequencing. The resulting mutational spectrum was compared with that derived from an examination of 322 spontaneous SUP4-o mutations selected in an isogenic wild-type (RAD1) strain. This comparison revealed that the rad1 mutator phenotype was associated with increases in the frequencies of single-base-pair substitution, single-base-pair deletion, and insertion of the yeast retrotransposon Ty. In the rad1 strain, the relative fractions of these events and their distributions within SUP4-o exhibited features similar to those for spontaneous mutagenesis in the isogenic RAD1 background. The increase in the frequency of Ty insertion argues that Ty transposition can be activated by unrepaired spontaneous DNA damage, which normally would be removed by excision repair. We discuss the possibilities that either translesion synthesis, a reduced fidelity of DNA replication, or a deficiency in mismatch correction might be responsible for the majority of single-base-pair events in the rad1 strain.

Base Composition↗

PubMed1995

Proteins that participate in nucleotide excision repair of DNA in mammalian cells.

The most versatile strategy for repair of damage to DNA, and the main process for repair of UV-induced damage, is nucleotide excision repair. In mammalian cells, the complete mechanism involves more than 20 polypeptides, and defects in many of these are associated with various forms of inherited disorders in humans. The syndrome xeroderma pigmentosum (XP) is associated with mutagen hypersensitivity and increased cancer frequency, and studies of the nucleotide excision repair defect in this disease have been particularly informative. Many of the XP proteins are now being characterized. XPA binds to DNA, with a preference for damaged base pairs. XPC activity is part of a protein complex with single-stranded DNA binding activity. The XPG protein is a nuclease.

Animals↗