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At least 19 recordsLinked to original sources

PubMed1979

Further characterization of a cell-free system for measuring replicative and repair DNA synthesis with cultured human fibroblasts and evidence for the involvement of DNA polymerase alpha in DNA repair.

DNA repair synthesis can be specifically measured in osmotically opened, confluent cultured human fibroblasts after exposure to DNA damaging agents such that both induction and mediation of DNA repair synthesis can take place in this cell-free system. Alternatively, by utilizing osmotically shocked, log phase cells and altering the DNA precursors, pH and ionic strength, replicative DNA synthesis can be specifically monitored. Autoradiographic studies show that virtually all of the nuclei from the lysates of the confluent, UV-iradiated cells are lightly labeled in the fashion characteristic of DNA repair. By contrast, only a fraction of nuclei is labeled in a population of unperturbed, opened log phase cells and the labeling is heavy and characteristic of replicative synthesis. Furthermore, equilibrium density gradient sedimentation shows that DNA synthesis in lysates of log-phase cells is semiconservative, whereas that with UV-irradiated cells is repair synthesis. This open cell system has been used to study the enzymology of DNA repair. Thus, dideoxythymidine triphosphate, a specific inhibitor of DNA polymerases beta and gamma, does not inhibit either replicative or repair synthesis. By contrast, aphidicolin, a specific inhibitor of DNA polymerase alpha, inhibits DNA repair and replicative synthesis in both intact and permeabilized cells. Finally, phage T4 UV-exonuclease stimulates repair synthesis, but only when phage T4 UV-endonuclease is also added to the UV-irradiated nuclei.

4-Nitroquinoline-1-oxide↗

PubMed1987

A comparative study of hepatic DNA repair, DNA replication and hepatotoxicity in the CD-1 mouse following multiple administrations of dimethylnitrosamine.

The objective of this study was to quantify hepatic DNA repair and DNA replication following multiple administrations of dimethylnitrosamine (DMN) and to determine if these events were correlated with hepatotoxicity. Male CD-1 mice, 50-100 days old, were dosed daily, p.o., with DMN in water at dose levels of 2, 4, 7 and 10 mg/kg for 2 weeks. After 2, 7 and 14 days of dosing, hepatocytes were isolated by an in situ perfusion procedure, incubated in the presence of [3H] thymidine, and fixed. Unscheduled as well as scheduled DNA synthesis were assessed by quantitative autoradiography. Unscheduled DNA synthesis (UDS) represents DNA repair while scheduled DNA synthesis (S phase) represents DNA replication. In addition, the animals' serum was examined for enzymes which indicate hepatic toxicity. After 1, 7 and 14 days of dosing, animals were orbital-bled and the serum was analyzed for serum glutamic pyruvic transaminase (SGPT), serum glutamic oxalacetic transaminase (SGOT), alkaline phosphatase (AP) and gamma-glutamyl transpeptidase (GGT). No morbidity or mortality was observed at dose levels of 2 and 4 mg/kg, but all animals receiving 7 and 10 mg/kg died after 4-6 days of dosing. GGT or AP were not elevated at any dose level or at any time point examined. At 4 mg/kg only a slight increase (less than or equal to 2 X) in the concentration of SGOT and SGPT was observed but a sharp increase (greater than 20 X) in replicative DNA synthesis was seen. The 2 mg/kg dose level of DMN did not increase replicative DNA synthesis and SGOT and SGPT were not elevated above control values at any time point following dosing at 2 mg/kg. A weakly positive DNA repair response was observed for dose levels of 4, 7 and 10 mg/kg DMN after two consecutive days of dosing. No DNA repair was observed after either 7 or 14 days of dosing at the 2 and 4 mg/kg/day levels. These results indicate that hepatic toxicity is associated with the induction of replicative DNA synthesis (S phase) but not with the induction of DNA repair. The results also confirm and extend a previous study (Doolittle et al., 1987b) indicating that a significant elevation in hepatic DNA replication is induced by hepatocarcinogens after multiple administrations of dose levels which do not alter hepatic DNA replication after a single administration. This finding indicates that the utility of the in vivo-in vitro hepatocyte assay may be enhanced by using a multi-dose protocol.

Animals↗

PubMedDate not supplied

Evaluation of the DNA-repair host-mediated assay. I. Induction of repairable DNA damage in E. coli cells recovered from liver, spleen, lungs, kidneys, and the blood stream of mice treated with methylating carcinogens.

The DNA-repair host-mediated assay was further calibrated by determining the genotoxic activities of 4 methylating carcinogens, namely, dimethylnitrosamine (DMNA), 1,2-dimethylhydrazine (SDMH), methyl nitrosourea (MNU) and methyl methanesulphonate (MMS) in various organs of treated mice. The ranking of the animal-mediated genotoxic activities of the compounds was compared with that obtained in DNA repair assays performed in vitro. The differential survival of strain E. coli K-12/343/113 and of its DNA-repair-deficient derivatives recA, polA and uvrB/recA, served as a measure of genotoxic potency. In the in vitro assays and at equimolar exposure concentrations, MMS and MNU are the most active chemicals, followed by DMNA, which shows a slight genotoxic effect only in the presence of mouse liver homogenate; SDMH has no activity under these conditions. In the host-mediated assays, the order of genotoxic potency of the compounds was quite different: those carcinogens which require mammalian metabolic activation, namely, DMNA and SDMH, show strong effects in liver and blood, a lesser effect in the lungs and kidneys and the least effect in the spleen. The activity of MNU, a directly acting compound, is similar in all organs investigated, but it is clearly lower than that of DMNA and SDMH. MMS, also a directly acting carcinogen, causes some (barely significant) effect at the highest dose tested. A similar order of potency was observed when the compounds were tested in intrasanguineous host-mediated assays with gene mutation as an endpoint. DMNA and SDMH induce comparable frequencies of L-valine-resistant mutants in E. coli K-12/343/113 recovered from liver and spleen of treated mice, the effect in the liver being the strongest. MNU is mutagenic only at a higher dose, while MMS shows no effect. The results are discussed with respect to the literature data on organ-specific DNA adduct formation induced by the compounds. It is concluded that qualitatively there is a good correlation between the degree of genotoxic activity found in the DNA repair host-mediated assay and DNA adduct formation in the animal's own cells. This is exemplified by the finding that the relative order of genotoxic activity of the 4 methylating agents in bacteria recovered from various organs (DMNA approximately equal to SDMH greater than MNU greater than MMS) is reflected by the same order of magnitude in DNA alkylation in corresponding mammalian organs. Quantitatively, the indirectly acting agents DMNA and SDMH seem to induce fewer genotoxic effects in bacteria present in the liver than would be expected on the basis of DNA-adduct formation data.

Alkylating Agents↗

PubMedDate not supplied

[The role of DNA repair processes in the repair of cytogenetic disorders. The participation of slow to repair DNA damage in the repair of cytogenetic disorders].

Caffeine was used to study the kinetics of cytogenetic damages repair in Chinese hamster fibroblasts. Its half-time (90 min) was shown to correlate with that of repair of slowly repaired DNA damages. The caffeine-induced increase in the number of irreparable DNA damages, attributed to inhibition of double-strand break repair, is in a quantitative correlation with the effect of the cytogenetic damage modification.

Animals↗

PubMed1997

Transfection enhancement in Bacillus subtilis displays features of a novel DNA repair pathway. II: Host constitutive expression, repair DNA synthesis, and in vitro activity.

In the Bacillus subtilis genetic system, transfection refers to uptake of isolated bacteriophage DNA by competent host cells, sometimes followed by productive cell infection. Previous studies have shown that ultraviolet (UV)-irradiation of the competent host cells, or cotransfection of UV-irradiated heterologous DNA, can increase the efficiency of transfection in some cases; these latter two phenomena have been called transfection enhancement (TE). In an accompanying paper, we show that TE is apparently confined to the B. subtilis phages that contain hydroxymethyluracil (HMU) in their DNA, and that the photoproduct in UV-irradiated DNA that mediates TE is specific, and different than the pyrimidine dimer, thymine glycol, uracil, or HMU. We also show that TE is due to reduced intracellular endonucleolytic attack of transfecting DNA. Based on this DNA base and nucleolytic specificity, we hypothesized that TE reflects the incidental action of a host DNA repair system on transfecting HMU phage DNA. In continuing these studies, we show here that duplex infecting HMU phage DNA is apparently inactivated by this same putative repair system when phage protein synthesis is blocked. We find, too, that this inactivation of infecting HMU phage DNA can be inhibited by UV-irradiated DNA, and that this process has a similar DNA base specificity as for TE. The survival of infecting HMU phage DNA is dependent on host DNA polymerase activity. We can detect specific DNA synthesis consistent with formation of repair patches when inactivation of infecting HMU phage DNA is ongoing, but not when it is inhibited by the presence of UV DNA or by allowing phage gene expression. Each of these results is consistent with the hypothesis that TE reflects the action of a novel DNA repair pathway. We show that a candidate TE-associated enzymatic activity can be detected in cell free extracts of uninfected, but not HMU phage-infected, B. subtilis cells. Correspondingly, the extracts of phage-infected cells appear to contain a diffusible factor that acts as an inhibitor of this host enzyme.

Bacillus subtilis↗

PubMed1994

DNA repair. DNA surveillance defect in cancer cells.

A defect in a protein involved in DNA-mismatch correction accounts for a common type of hereditary colon cancer, adding to the evidence that DNA repair is central to counteracting the transformation of human cells.

Adenosine Triphosphatases↗

PubMed2006

DNA repair: DNA polymerase zeta and Rev1 break in.

DNA polymerase zeta and Rev1 play key roles in replication past DNA lesions. New work shows that the yeast checkpoint kinase Mec1 recruits a complex consisting of polymerase zeta and Rev1 to DNA double-strand breaks. This study highlights the role of polymerases that mediate translesion synthesis in the response to DNA double-strand breaks.

DNA Repair↗

PubMedDate not supplied

DNA repair, DNA synthesis and cell cycle delay in human lymphoblastoid cells differentially sensitive to the cytotoxic effects of nitrogen mustard.

Two cloned human lymphoblastoid cell lines, Raji and TK6, differ in their sensitivity to the cytotoxic effects of nitrogen mustard (HN2). Raji cells exhibit a biphasic response with an initial D value of 0.06 microgram/ml and a final slope of 0.25 microgram/ml. TK6 cells were considerably more sensitive, D0 value 0.02 microgram/ml. Dose-response relationships for delay in cell cycle progression were measured using flow cytometry. Delay in S-phase traverse was concentration-dependent in both cell lines, and at a given concentration was 2-fold greater in TK6 than in Raji. Numbers of crosslinks (determined by alkaline elution) increased linearly with increasing HN2 concentration and were approximately 2-fold higher in TK6 than in Raji. At equal levels of DNA crosslinks, rates of removal were similar in both cell lines. Inhibition of [3H]TdR uptake was concentration-dependent and the extent of inhibition was similar in both cell lines. Recovery from HN2-induced inhibition of cell cycle progression markedly preceded recovery from inhibition of [3H]TdR incorporation suggesting that nucleotide pools are markedly perturbed in HN2-treated cells. The difference in sensitivity of these two cell lines cannot be adequately explained by differences in amounts of initial DNA damage, rates of repair, differential S-phase delay or rate of loss of DNA crosslinks.

Burkitt Lymphoma↗

PubMed1991

The mode of action of 1-beta-D-arabinofuranosylcytosine in inhibiting DNA repair; new evidence using a sensitive assay for repair DNA synthesis and ligation in permeable cells.

Mammalian cells permeabilised by treatment with saponin are capable of UV excision repair. We have developed an assay system which permits measurement of the later stages of repair, i.e. repair synthesis and ligation. Incomplete repair sites are accumulated in UV-irradiated cells by incubating them with DNA synthesis inhibitors hydroxyurea and aphidicolin. On removal of the inhibitors at the time of permeabilisation, these incomplete sites, detected as DNA breaks, are rapidly ligated in a reaction that requires deoxyribonucleoside triphosphates and is blocked by aphidicolin. Thus ligation is possible only after a significant amount of DNA synthesis. We have used the assay to clarify the mode of inhibition of DNA repair by 1-beta-D-arabinofuranosylcytosine (ara C), another DNA polymerase inhibitor. It is well known that incomplete repair sites accumulated in whole cells with ara C are ligated at a slow rate, if at all. The hypothesis that ara C blocks or reduces further polymerisation after its incorporation into repair patches is disproved by our demonstration that, in permeable cells, the accumulated DNA breaks are ligated very rapidly. The likely explanation of the action of ara C is that, once phosphorylated, it remains in the cell as ara CTP and continues to inhibit polymerisation through competition with dCTP; in permeable cells, it readily leaks out.

Aphidicolin↗

PubMed1999

DNA repair, DNA replication, and UV mutagenesis.

Cells that have been irradiated with ultraviolet light (UV) suffer damage to their DNA, primarily in the form of covalent linkage between adjacent pyrimidines. Such photoproducts represent blocks to RNA and DNA polymerases and are potentially mutagenic. Blockage of RNA polymerase II by a photoproduct in the transcribed strand of an active gene leads to induction of the p53 protein, which induces pleiotropic responses that may include apoptotic cell death. If a cell survives, the blocked polymerase targets the nucleotide excision repair machinery to the site of the lesion, which is repaired in an error-free manner. Repair coupled to transcription in this manner strongly influences the mutation spectrum induced by UV, reducing the proportion of base substitutions that arise from photoproducts on the transcribed strand. If the damage persists when the DNA is replicated in S-phase, either because the cell is unable to repair the damage or because there is insufficient time between the induction of damage and the onset of S-phase. To do so, the replicative DNA polymerase complex may be blocked. In this situation, lesion bypass can be accomplished using an error-free mechanism, or using an error-prone mechanism that involves the newly described, non-processive DNA polymerase zeta encoded by the human homolog of the yeast REV3 gene.

Animals↗

PubMed2017

Small-Molecule Inhibitors Targeting DNA Repair and DNA Repair Deficiency in Research and Cancer Therapy.

To maintain stable genomes and to avoid cancer and aging, cells need to repair a multitude of deleterious DNA lesions, which arise constantly in every cell. Processes that support genome integrity in normal cells, however, allow cancer cells to develop resistance to radiation and DNA-damaging chemotherapeutics. Chemical inhibition of the key DNA repair proteins and pharmacologically induced synthetic lethality have become instrumental in both dissecting the complex DNA repair networks and as promising anticancer agents. The difficulty in capitalizing on synthetically lethal interactions in cancer cells is that many potential targets do not possess well-defined small-molecule binding determinates. In this review, we discuss several successful campaigns to identify and leverage small-molecule inhibitors of the DNA repair proteins, from PARP1, a paradigm case for clinically successful small-molecule inhibitors, to coveted new targets, such as RAD51 recombinase, RAD52 DNA repair protein, MRE11 nuclease, and WRN DNA helicase.

DNA Helicases↗

PubMed1995

Replication protein A binds to regulatory elements in yeast DNA repair and DNA metabolism genes.

Saccharomyces cerevisiae responds to DNA damage by arresting cell cycle progression (thereby preventing the replication and segregation of damaged chromosomes) and by inducing the expression of numerous genes, some of which are involved in DNA repair, DNA replication, and DNA metabolism. Induction of the S. cerevisiae 3-methyladenine DNA glycosylase repair gene (MAG) by DNA-damaging agents requires one upstream activating sequence (UAS) and two upstream repressing sequences (URS1 and URS2) in the MAG promoter. Sequences similar to the MAG URS elements are present in at least 11 other S. cerevisiae DNA repair and metabolism genes. Replication protein A (Rpa) is known as a single-stranded-DNA-binding protein that is involved in the initiation and elongation steps of DNA replication, nucleotide excision repair, and homologous recombination. We now show that the MAG URS1 and URS2 elements form similar double-stranded, sequence-specific, DNA-protein complexes and that both complexes contain Rpa. Moreover, Rpa appears to bind the MAG URS1-like elements found upstream of 11 other DNA repair and DNA metabolism genes. These results lead us to hypothesize that Rpa may be involved in the regulation of a number of DNA repair and DNA metabolism genes.

Base Sequence↗

PubMed2005

Saccharomyces cerevisiae histone H2A Ser122 facilitates DNA repair.

DNA repair takes place in the context of chromatin. Recently, it has become apparent that proteins that make up and modulate chromatin structure are involved in the detection and repair of DNA lesions. We previously demonstrated that Ser129 in the carboxyl-terminal tail of yeast histone H2A is important for double-strand-break responses. By undertaking a systematic site-directed mutagenesis approach, we identified another histone H2A serine residue (Ser122) that is important for survival in the presence of DNA-damaging agents. We show that mutation of this residue does not affect DNA damage-dependent Rad53 phosphorylation or G(2)/M checkpoint responses. Interestingly, we find that yeast lacking H2A S122 are defective in their ability to sporulate. Finally, we demonstrate that H2A S122 provides a function distinct from that of H2A S129. These data demonstrate a role for H2A S122 in facilitating survival in the presence of DNA damage and suggest a potential role in mediating homologous recombination. The distinct roles of H2A S122 and S129 in mediating these responses suggest that chromatin components can provide specialized functions for distinct DNA repair and survival mechanisms and point toward the possibility of a complex DNA damage responsive histone code.

Amino Acid Sequence↗

PubMed2005

Human MMS21/NSE2 is a SUMO ligase required for DNA repair.

DNA repair is required for the genomic stability and well-being of an organism. In yeasts, a multisubunit complex consisting of SMC5, SMC6, MMS21/NSE2, and other non-SMC proteins is required for DNA repair through homologous recombination. The yeast MMS21 protein is a SUMO ligase. Here we show that the human homolog of MMS21 is also a SUMO ligase. hMMS21 stimulates sumoylation of hSMC6 and the DNA repair protein TRAX. Depletion of hMMS21 by RNA interference (RNAi) sensitizes HeLa cells toward DNA damage-induced apoptosis. Ectopic expression of wild-type hMMS21, but not its ligase-inactive mutant, rescues this hypersensitivity of hMMS21-RNAi cells. ATM/ATR are hyperactivated in hMMS21-RNAi cells upon DNA damage. Consistently, hMMS21-RNAi cells show an increased number of phospho-CHK2 foci. Finally, we show that hMMS21-RNAi cells show a decreased capacity to repair DNA lesions as measured by the comet assay. Our findings suggest that the human SMC5/6 complex and the SUMO ligase activity of hMMS21 are required for the prevention of DNA damage-induced apoptosis by facilitating DNA repair in human cells.

Amino Acid Sequence↗

PubMed1992

Cis and trans mechanisms of DNA repair.

DNA repair is essential for genetic stability and variability. Remarkable advances in the understanding of DNA repair by the molecular analysis of the substrate (gene repair) or the enzyme (repair genes), emphasize evolutionary conservation. Recent progress also stresses the interaction(s) between DNA repair and numerous other cellular metabolic processes, including non-nuclear and/or non-genetic responses.

Animals↗

PubMed2006

Association of DNA repair polymorphisms with DNA repair functional outcomes in healthy human subjects.

We investigated association between polymorphisms in DNA repair genes and the capacity to repair DNA damage induced by gamma-irradiation and by base oxidation in a healthy population. Irradiation-specific DNA repair rates were significantly decreased in individuals with XRCC1 Arg399Gln homozygous variant genotype (0.45 +/- 0.47 SSB/10(9) Da) than in those with wild-type genotype (1.10 +/- 0.70 SSB/10(9) Da, P=0.0006, Mann-Witney U-test). The capacity to repair oxidative DNA damage was significantly decreased among individuals with hOGG1 Ser326Cys homozygous variant genotype (0.37 +/- 0.28 SSB/10(9) Da) compared to those with wild-type genotype (0.83 +/- 0.79 SSB/10(9) Da, P=0.008, Mann-Witney U-test). Investigation of genotype combinations showed that the increasing number of variant alleles for both XRCC1 Arg399Gln and APE1 Asn148Glu polymorphisms resulted in a significant decrease of irradiation-specific repair rates (P=0.008, Kruskal-Wallis test). Irradiation-specific DNA repair rates also decreased with increasing number of variant alleles in XRCC1 Arg399Gln in combination with variant alleles for two other XRCC1 polymorphisms, Arg194Trp and Arg280His (P=0.002 and P=0.005, respectively; Kruskal-Wallis test). In a binary combination variant alleles of hOGG1 Ser326Cys and APE1 Asn148Glu polymorphisms were associated with a significant decrease in the capacity to repair DNA oxidative damage (P=0.018, Kruskal-Wallis test). In summary, XRCC1 Arg399Gln and hOGG1 Ser326Cys polymorphisms seem to exert the predominant modulating effect on irradiation-specific DNA repair capacity and the capacity to repair DNA oxidative damage, respectively.

Adult↗

PubMed1995

Current sample handling methods for measurement of platinum-DNA adducts in leucocytes in man lead to discrepant results in DNA adduct levels and DNA repair.

DNA adduct levels were measured with atomic spectroscopy in white blood cells (WBCs) from patients with solid tumours who were treated with six weekly courses of cisplatin. In 21 patients (I) the WBCs were collected after thawing frozen whole-blood samples according to a previously described method. In 32 other patients (II) WBCs were collected immediately after blood sample collection. The two methods for WBC collection were also compared in vitro. The maximal DNA adduct levels in vivo after the first course were in I 2.48 +/- 1.14 and in II 1.28 +/- 0.40 pg of platinum per microgram of DNA (P < 0.0001). The DNA 'repair' in the first course (DNA adduct level at the end of the infusion minus the level 15 h post infusion) was in I 40% +/- 29% and in II 18% +/- 29% (P = 0.009). These differences were consistent in all measured courses. In vitro, the DNA adduct levels in the freshly prepared WBCs were significantly lower at 0, 1 and 4, but not 24 h, after start of the incubation with cisplatin than in the WBCs collected after freezing and thawing the blood sample. The same experiment with carboplatin in vitro also resulted in significantly lower adducts in freshly isolated WBCs. The higher DNA adduct levels and DNA 'repair' in I are caused by remaining unbound cisplatin in the sample tubes, which can form DNA adducts ex vivo. The same results in vivo can be anticipated when carboplatin is used.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed1989

The effect of aging and dietary restriction on DNA repair.

DNA repair was studied as a function of age in cells isolated from both the liver and the kidney of male Fischer F344 rats. DNA repair was measured by quantifying unscheduled DNA synthesis induced by UV irradiation. Unscheduled DNA synthesis decreased approximately 50% between the ages of 5 and 30 months in both hepatocytes and kidney cells. The age-related decline in unscheduled DNA synthesis in cells isolated from the liver and kidney was compared in rats fed ad libitum and rats fed a calorie-restricted diet; calorie restriction has been shown to increase the survival of rodents. The level of unscheduled DNA synthesis was significantly higher in hepatocytes and kidney cells isolated from the rats fed the restricted diet. Thus, calorie restriction appears to retard the age-related decline in DNA repair.

Aging↗