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PubMed1999

Direct involvement of p53 in the base excision repair pathway of the DNA repair machinery.

The p53 tumor suppressor that plays a central role in the cellular response to genotoxic stress was suggested to be associated with the DNA repair machinery which mostly involves nucleotide excision repair (NER). In the present study we show for the first time that p53 is also directly involved in base excision repair (BER). These experiments were performed with p53 temperature-sensitive (ts) mutants that were previously studied in in vivo experimental models. We report here that p53 ts mutants can also acquire wild-type activity under in vitro conditions. Using ts mutants of murine and human origin, it was observed that cell extracts overexpressing p53 exhibited an augmented BER activity measured in an in vitro assay. Depletion of p53 from the nuclear extracts abolished this enhanced activity. Together, this suggests that p53 is involved in more than one DNA repair pathway.

Animals↗
PubMed1989

Failure to induce a DNA repair gene, RAD54, in Saccharomyces cerevisiae does not affect DNA repair or recombination phenotypes.

The Saccharomyces cerevisiae RAD54 gene is transcriptionally regulated by a broad spectrum of DNA-damaging agents. Induction of RAD54 by DNA-damaging agents is under positive control. Sequences responsible for DNA damage induction (the DRS element) lie within a 29-base-pair region from -99 to -70 from the most proximal transcription start site. This inducible promoter element is functionally separable from a poly(dA-dT) region immediately downstream which is required for constitutive expression. Deletions which eliminate induction of RAD54 transcription by DNA damage but do not affect constitutive expression have no effect on growth or survival of noninducible strains relative to wild-type strains in the presence of DNA-damaging agents. The DRS element is also not required for homothallic mating type switching, transcriptional induction of RAD54 during meiosis, meiotic recombination, or spontaneous or X-ray-induced mitotic recombination. We find no phenotype for a lack of induction of RAD54 message via the damage-inducible DRS, which raises significant questions about the physiology of DNA damage induction in S. cerevisiae.

Base Sequence↗
PubMed2006

Differential recruitment of DNA Ligase I and III to DNA repair sites.

DNA ligation is an essential step in DNA replication, repair and recombination. Mammalian cells contain three DNA Ligases that are not interchangeable although they use the same catalytic reaction mechanism. To compare the recruitment of the three eukaryotic DNA Ligases to repair sites in vivo we introduced DNA lesions in human cells by laser microirradiation. Time lapse microscopy of fluorescently tagged proteins showed that DNA Ligase III accumulated at microirradiated sites before DNA Ligase I, whereas we could detect only a faint accumulation of DNA Ligase IV. Recruitment of DNA Ligase I and III to repair sites was cell cycle independent. Mutational analysis and binding studies revealed that DNA Ligase I was recruited to DNA repair sites by interaction with PCNA while DNA Ligase III was recruited via its BRCT domain mediated interaction with XRCC1. Selective recruitment of specialized DNA Ligases may have evolved to accommodate the particular requirements of different repair pathways and may thus enhance efficiency of DNA repair.

Animals↗
PubMed2005

The emerging role of DNA repair proteins as predictive, prognostic and therapeutic targets in cancer.

Advanced cancer is the second leading cause of death in the western world. Chemotherapy and radiation are the two main treatment modalities currently available to improve patient outcomes, but treatment related toxicity and the emergence of resistance limit their effectiveness. Hence there is an urgent need to develop novel treatment strategies. Rapid advances in cancer biology have identified key pathways involved in the repair of DNA damage induced by chemotherapeutic agents and irradiation. Efficient DNA repair in the cancer cell is an important mechanism for therapeutic resistance. Up to 130 genes have been identified that are associated with human DNA repair. Several of these proteins are emerging as important predictive and prognostic factors in solid tumours. Inhibition of DNA repair has the potential to enhance the efficacy of currently available DNA damaging agents. In recent years, several promising drug targets have been identified and novel drugs synthesised that target specific DNA repair proteins. These agents have shown impressive anti-cancer effects in preclinical studies in combination with chemotherapy or irradiation. Their role in human cancer is now being investigated in early phase clinical trials in combination with chemotherapy. MGMT inhibitors, PARP inhibitors and methoxyamine are currently in early stages of clinical development. Innovative clinical trial designs are essential to evaluate the potential of DNA repair inhibitor in cancer therapy.

Animals↗
PubMed1975

Carcinogen-induced DNA repair in nucleotide-permeable Escherichia coli cells. Induction of DNA repair by the carcinogens methyl and ethyl nitrosourea and methyl methanesulfonate.

Ether-permeabilized (nucleotide-permeable) cells of Escherichia coli show excision repair of their DNA after having been exposed to the carcinogens N-methyl-N-nitrosourea (MeNOUr), N-ethyl-N-nitrosourea (EtNOUr) and methyl methanesulfonate (MeSO2OMe) which are known to bind covalently to DNA. Defect mutations in genes uvrA, uvrB, uvrC, recA, recB, recC and rep did not inhibit this excision repair. Enzymic activities involved in this repair were identified by measuring size reduction of DNA, DNA degradation to acid-soluble nucleotides and repair polymerization. 1. In permeabilized cells methyl and ethyl nitrosourea induced endonucleolytic cleavage of endogenous DNA, as determined by size reduction of denatured DNA in neutral and alkaline sucrose gradients. An enzymic activity from E. coli K-12 cell extracts was purified (greater than 2000-fold) and was found to cleave preferentially methyl-nitrosourea-treated DNA and to convert the methylated supercoiled DNA duplex (RFI) of phage phiX 174 into the nicked circular form. 2. Degradation of alkylated cellular DNA to acid solubility was diminished in a mutant lacking the 5' leads to 3' exonucleolytic activity of DNA polymerase I but was not affected in a mutant which lacked the DNA polymerizing but retained the 5' leads 3' exonucleolytic activity of DNA polymerase I. 3. An easily measurable effect is carcinogen-induced repair polymerization, making it suitable for detection of covalent binding of carcinogens and potentially carcinogenic compounds.

Carcinogens↗
PubMed2007

Expression of somatic DNA repair genes in human testes.

Meiosis is the key process for recombination and reduction of the diploid chromosome set to a haploid one. Many genes that have been found in yeast or mouse models to play a role in meiosis are also important for the repair of DNA damage in somatic cells. To study the DNA repair gene transcriptome during male germ cell development, we have developed a specialized cDNA microarray with 181 human genes which are involved in different somatic DNA repair pathways and/or cell cycle control and 45 control house-keeping genes. This DNA repair gene chip was used to quantify the mRNA expression levels in three human testes samples versus a fibroblast RNA pool. Two hundred twenty genes on the chip (including house-keeping genes) showed detectable expression levels in adult testes. Sixty-four DNA repair- and cell cycle-associated genes showed higher expression levels in testicular cells than in mitotically dividing fibroblasts and, therefore, are likely to be implicated in meiosis. The microarray results of 17 genes with increased expression levels were validated with reverse Northern blots or real-time quantitative RT PCR. Systematic analyses of the meiotic DNA repair gene transcriptome may provide new insights into the genetics of male (in)fertility.

Biomarkers↗
PubMed2005

A FancD2-monoubiquitin fusion reveals hidden functions of Fanconi anemia core complex in DNA repair.

In DNA damage responses, the Fanconi anemia (FA) protein, FancD2, is targeted to chromatin and forms nuclear foci following its monoubiquitination, a process likely catalyzed by the FA core complex. Here, we show that a chicken FancD2-ubiquitin fusion protein, carrying a Lys-Arg substitution removing the natural monoubiquitination site (D2KR-Ub), could reverse cisplatin hypersensitivity and localize to chromatin in FANCD2-deficient DT40 cells. Importantly, the chromatin targeting was dependent on three core complex components as well as the hydrophobic surface of ubiquitin that may direct protein-protein interactions. Furthermore, a constitutively chromatin bound fusion of D2KR-histone H2B could complement cisplatin sensitivity in FANCD2- but not FANCC-, FANCG-, or FANCL-deficient cells. Thus these core complex components have an additional function in the DNA repair, which is independent of the monoubiquitination and chromatin targeting of FancD2. These results define functional consequences of FancD2 monoubiquitination and reveal previously hidden functions for the FA protein core complex.

Animals↗
PubMed2005

Genetic polymorphisms and possible gene-gene interactions in metabolic and DNA repair genes: effects on DNA damage.

We investigated in a central European population, the association between genetic polymorphisms in several genes coding for xenobiotic metabolizing enzymes (CYP1A1, CYP2E1, EPHX1, GSTP1, GSTM1 and GSTT1) and in DNA repair genes (XPD, XPG, XPC and XRCC1) and the levels of single-strand breaks (SSBs) and SSB endonuclease III sensitive sites (endoIII sites) in peripheral blood lymphocytes. No significant differences in the mean levels of SSBs and endoIII sites after stratification for main confounders and occupational exposure were observed in the studied population. Significantly higher levels of SSBs were observed in individuals bearing the wild-type alleles (AA) (0.75+/-0.51SSB/10(9)Da) and heterozygous (AC) genotypes (0.67+/-0.49SSB/10(9)Da) compared to those with homozygous XPD (CC) genotype (0.43+/-0.28SSB/10(9)Da, P=0.033). A moderate increase in the levels of SSBs was also found in individuals with the homozygous XPG exon 15 wild type (GG) and heterozygous (GC) genotypes in comparison to those with the homozygous (CC) genotype (P=0.066) and in individuals with low activity EPHX1 genotype in comparison to those with high activity genotype. Nevertheless, these differences were not statistically significant. No other significant association was found. When gene-gene interactions were evaluated, a combination of EPHX1 activity genotypes with that of either XPD or XPG significantly (P=0.003 and 0.016, respectively) modulated SSB levels resulting in a three-fold difference between the "protective" and the "adverse" genotype-combinations. Almost three-fold differences in SSB levels were found between the "protective" and the "adverse" genotype-combinations of EPHX1 activity genotype and GSTM1 or GSTT1 genotypes, respectively. In conclusion, our results suggest a relation between markers of genotoxicity and polymorphisms in genes coding for xenobiotic metabolizing and DNA repair enzymes as well as a modulating effect of combinations of these polymorphisms.

Adult↗
PubMedDate not supplied

[Irradiation as a test-factor revealing dark DNA repair capacity of seeds].

Dark repair of DNA was studied in embryos excised from the advanced sugar beet seeds. Significant increase (from 19 to 321%) in the level of dark DNA repair has been established for all studied conditions of the advanced treatment. Acute -irradiation has been used to investigate the ability of advanced seeds to repair additional DNA damages caused by a standard irradiation dose. It has been concluded that irradiation factor allows to test capacity of DNA repair systems. The later, we suggest, can be used to define the optimal conditions for the seed advancement.

Beta vulgaris↗
PubMed1987

The endogenous nuclease sensitivity of repaired DNA in human fibroblasts.

The limited DNA excision repair that occurs in the chromatin of UV-irradiated growth arrested cells isolated from a xeroderma pigmentosum (XP) complementation group C patient is clustered in localized regions. The repaired DNA was found to be more sensitive to nicking by endogenous nucleases than the bulk of the DNA. The extra-sensitivity does not change with increasing amounts of DNA damage or repair activity in the locally-repaired regions and is retained through a 24-h chase period. We suggest that these results are due to the occurrence of DNA repair limited to pre-existing, non-transient chromatin fractions that contain actively transcribed DNA. A similar extra-sensitivity of repaired DNA was not detected in cells of normal or XP complementation group A strains that exhibit either normal or limited repair located randomly throughout their genomes. The association between endogenous nuclease sensitivity and clustered repair probably defines a normal excision repair pathway that is specific for selected chromatin domains. The repair defect in XP-C strains may be one in pathways targeted for other endogenous nuclease-resistant domains.

Cell Nucleus↗
PubMed2005

Late activation of stress kinases (SAPK/JNK) by genotoxins requires the DNA repair proteins DNA-PKcs and CSB.

Although genotoxic agents are powerful inducers of stress kinases (SAPK/JNK), the contribution of DNA damage itself to this response is unknown. Therefore, SAPK/JNK activation of cells harboring specific defects in DNA damage-recognition mechanisms was studied. Dual phosphorylation of SAPK/JNK by the genotoxin methyl methanesulfonate (MMS) occurred in two waves. The early response (< or = 2 h after exposure) was similar in cells knockout for ATM, PARP, p53, and CSB or defective in DNA-PK(cs) compared with wild-type cells. The late response however (> or = 4 h), was drastically reduced in DNA-PK(cs) and Cockayne's syndrome B (CSB)-deficient cells. Similar results were obtained with human cells lacking DNA-PK(cs) and CSB. Activation of SAPK/JNK by MMS was not affected upon inhibition of base excision repair (BER), indicating base damage itself does not signal to SAPK/JNK. Because SAPK/JNK activation was attenuated in nongrowing cells, DNA replication-dependent processing of lesions, involving DNA-PK(cs) and CSB, appears to be required. DNA-PK(cs) coprecipitates with SEK1/MKK4 and SAPK/JNK, supporting a role of DNA-PK(cs) in SAPK/JNK activation. In this process, Rho GTPases are involved since inhibition of Rho impairs MMS-induced signaling to SAPK/JNK. The data show that sensing of DNA damage by DNA-PK(cs) and CSB causes a delayed SEK1/MKK4-mediated dual phosphorylation of SAPK/JNK.

Alkylation↗
PubMed1976

Carcinogen-induced DNA repair in nucleotide-permeable Escherichia coli cells. Analysis of DNA repair induced by the carcinogens N-acetoxy-N-2-acetylaminofluorene and 7-bromomethyl-benz(a)anthracene.

Upon exposure to the carcinogens N-acetoxy-N-2-acetylaminofluorene and 7-bromomethyl-benz[a]anthracene, which bind covalently to DNA, ether-permeabilized (nucleotide-permeable) Escherichia coli wild-type cells responded with DNA excision repair. This repair was missing in mutants carrying defects in genes uvrA, uvrB and uvrC, whereas it was present in uvrD and several rec mutants. Enzymic activities involved were identified by measuring repair polymerization and size reduction of denatured DNA. 1. An easily measurable effect in E. coli wild-type cells was carcinogen-induced repair polymerization. When initiated by N-acetoxy-N-2-acetylaminofluorene or 7-bromomethyl-benz[a]anthracene, it depended upon an ATP-requiring step; CTP, GTP or UTP did not substitute for ATP. DNA repair synthesis was inhibited by p-chloromercuribenzoate and quinacrine. In uvrA, uvrB and uvrC mutants no carcinogen-stimulated DNA synthesis could be detected, indicating that steps involved in pyrimidine dimer excision are also involved in chemorepair. In recA, recB and recC mutant cells, repair synthesis was stimulated by the carcinogens to a normal extent. This evidence excludes the ATP-dependent recB,C deoxyribonuclease and recA gene products as playing an important role in carcinogen-induced excision repair. polA1 cells showed drastically reduced levels of rapair polymerization, indicating that DNA polymerase I is the main polymerizing enzyme. 2. As determined by DNA size reduction in alkaline sucrose gradients, the arylalkylating carcinogens caused endonucleolytic cleavage of endogenous DNA in wild-type cells. This incision step was most effectively performed in the presence of ATP; UTP, CTP and GTP were only slightly effective. Incision was inhibited by p-chloromercuribenzoate and quinacrine. When exposed to the arylalkylating carcinogens, uvrA, uvrB and uvrC mutant cells did not perform the incision step in the presence of ATP, suggesting the involvement of the respective gene products in the initiation of chemorepair.

Acetoxyacetylaminofluorene↗
PubMed2004

Epstein-Barr virus latent membrane protein 1 induces micronucleus formation, represses DNA repair and enhances sensitivity to DNA-damaging agents in human epithelial cells.

The latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is a viral oncogene and it is essential for the transformation of resting B cells by the virus. The protein acts as a ligand-less membrane receptor and triggers numerous cellular signaling pathways. Cellular transformation frequently has been associated with genomic instability. To investigate whether EBV LMP1 induces chromosomal aberrations, micronucleus (MN) formation was examined in LMP1-expressing epithelial cells. The expression of wild-type LMP1 enhanced both spontaneous and bleomycin-induced MN formation. MN formation may be induced by inactivation of DNA repair and, therefore, we investigated the effect of LMP1 on DNA repair, using a host cell reactivation (HCR) assay. In the HCR assay, LMP1 reduced the capacity for DNA repair of both NPC-TW01 (p53-wild-type) and H1299 (p53-deficient) cells. As reduction of DNA repair by LMP1 occurs in p53-wild-type and p53-deficient cells, it seems that LMP1 can repress DNA repair in a p53-independent manner. Inactivation of DNA repair may render cells sensitive to DNA-damaging agents. In this study, H1299 cells harboring LMP1 were shown to be more sensitive to UV and bleomycin than those with a vector control. Using various deletion mutants of EBV LMP1 to determine the regions of LMP1 required to enhance MN formation, inhibit DNA repair and sensitize cells to DNA-damaging agents, we found that the region a. a. 189-222 (located within the CTAR1 domain) was responsible for sensitizing cells to UV and bleomycin, as well as for enhancing MN formation and repressing DNA repair. Based on these results, we suggest that disruption of DNA repair by LMP-1 results in an accumulation of unrepaired DNA and consequent genomic instability, which may contribute to the oncogenesis of LMP1 in human epithelial cells.

Adenocarcinoma↗
PubMed2003

Defining the function of XPC protein in psoralen and cisplatin-mediated DNA repair and mutagenesis.

DNA damage recognition plays an important role in DNA repair and mutagenesis. Failure to recognize DNA damage may lead to DNA replication without damage repair as well as mutation accumulation. Mutations can lead to many disease conditions. XPC is a DNA damage recognition protein that binds to damaged DNA templates at a very early stage during the DNA repair process. We have studied the role of the XPC protein in DNA cross-link reagents, psoralen and cisplatin, mediated DNA repair and mutagenesis. When psoralen and cisplatin-damaged plasmid DNA was transfected into xeroderma pigmentosum group C (XPC) cells, which were defective in the XPC gene, very distinct mutation frequency and spectrum was observed: a decreased mutation frequency for psoralen-damaged plasmid and an increased mutation frequency for cisplatin-damaged plasmid; in contrast, most mutations generated by psoralen in XPC cells were T-to-G transversions and most mutations generated by cisplatin in XPC cells were large deletions. We also determined the DNA repair ability of XPC cells by both host cell reactivation (HCR) assay and in vitro DNA repair assay. The HCR results showed greatly reduced host cell reactivation of a luciferase reporter for both psoralen and cisplatin-damaged plasmid DNA in XPC cells. The in vitro DNA repair results revealed a defective repair capacity for both psoralen and cisplatin-damaged plasmid DNA in nuclear extract prepared from XPC cells. However, this defective DNA repair activity was partially restored when a functional XPC protein was supplemented into the XPC nuclear extract prior to the reaction. These results suggest that the XPC protein DNA damage recognition function plays a crucial role in DNA repair initiation and mutation avoidance and XPC defects may lead to increased mutations and high risk for disease progression.

Base Sequence↗
PubMed1992

[Effect of hematoporphyrin derivative (HPD) plus light on DNA repair synthesis and enhancement of HPD photosensitization by vitamin C in mouse hepatoma].

There are only a few reports in the literature on photodynamic effects of HPD on gene expression and the repair of DNA damage. Previously, we suggested that nuclear transcription activity was markedly suppressed by HPD plus light. In this study, we investigated the photodynamic effect or DNA repair synthesis as measured by unscheduled DNA synthesis (UDS) in hepatoma cells. The results indicated that DNA repair capacity was greatly inhibited by HPD plus light. When hepatoma cells were pretreated with HPD of different concentrations and then exposed to the same light dose, significant inhibitions of UDS over control were seen and appeared in a dose-dependent fashion. In addition, while hepatoma cells following the exposure to HPD of the same concentration were irradiated with different time, the UDS was markedly decreased as the duration of irradiation was prolonged. However, no changes were observed when either HPD or light was used alone. The present study also included an experiment to determine whether HPD combined with vitamin C gives an enhanced photosensitization. It was found that vitamin C significantly potentiated the suppressing effect on DNA repair synthesis.

Animals↗