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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↗
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↗
PubMed2025

WDFY2 promotes MRN complex formation required for homologous recombination-mediated DNA repair.

The MRE11-RAD50-NBS1 (MRN) complex is fundamental for detecting and repairing DNA double-strand breaks (DSBs), thereby safeguarding genome integrity. However, the precise mechanism governing MRN complex recruitment to DSBs remains largely unexplored. Here, we identify WD40- and FYVE domain-containing protein 2 (WDFY2) as an important regulator of MRN complex formation at DNA damage sites, facilitating homologous recombination (HR) repair. Mechanistically, WDFY2 is phosphorylated at serine 84 by the ATM-CHK2 axis, priming it for recruitment to DSBs. Through direct interactions with MRE11 and NBS1, WDFY2 bridges the MRE11-RAD50 subcomplex with NBS1, thereby promoting MRN complex formation at DSBs and DNA end resection. WDFY2 deficiency, as well as the non-phosphorylatable S84A mutant, results in impaired HR repair and reduced cell survival following DNA damage. Collectively, our findings establish WDFY2 as a key platform for MRN complex loading at DSBs and HR repair, highlighting it as a potential therapeutic target for cancer treatment.

Humans↗
PubMed1978

Carcinogen-induced DNA repair in nucleotide-permeable Escherichia coli cells. Analysis of DNA repair induced by carcinogenic K-region epoxides and 1,2,3,4-diepoxybutane.

Ether-permeabilized (nucleotide-permeable) Escherichia coli cells exhibited DNA excision repair when exposed to the following carcinogenic K-region epoxides: 7-methyl- and 7,12-dimethyl-benz[a]anthracene-5,6-oxide, chrysene-5,6-oxide and benzo[a]pyrene-4,5-oxide. This DNA excision repair was missing in uvr A and uvr B mutant cells. The K-region epoxide phenanthrene-9,10-oxide was ineffective in all E. coli strains tested. In contrast to the K-region epoxides which where found active only in wild type cells, 1,2,3,4-diepoxybutane and the 6,7-epoxides of the tumor promoter TPA (12-O-tetradecanoyl-phorbol-13-acetate) elicited DNA repair in uvrA, uvrB mutant cells as well. Enzymic activities catalyzing particular repair steps were identified by determining a) repair polymerization and b) size reduction of denatured DNA. A) An easily quantifiable effect in E. coli wild type cells was epoxide-induced repair polymerization. None of the K-region epoxides tested stimulated DNA repair synthesis in uvrA, uvrB mutant cells, indicating that the uvrA-, uvrB-controlled UV-endonuclease initiated excision repair by cleaving epoxide-damaged DNA. 1,2,3,4-Diepoxybutane and the TPA-6,7-oxides induced DNA repair polymerization in uvr-deficient cells, although to a lesser extent than in wild type cells, suggesting the involvement of uvr-independent incision steps. None of the epoxides induced repair polymerization in a mutant (polA107) lacking the 5'--3'exonucleolytic activity of DNA polymerase I (exonuclease VI). The absence of any repair polymerization in the polA107 mutant indicates that the exonuclease VI plays a central role in removing epoxide-damaged nucleotides. As evidenced by greatly reduced levels of repair polymerization measured in polA1 cells, DNA polymerase I was the main polymerizing enzyme. b) As a consequence of treatment with 7-methyl-benz[a]anthracene-5,6-oxide, DNA from wild type cells, contrary to uvrA mutant cells, showed size reduction after denaturation and sedimentation in alkaline sucrose gradients. This is explained by repair-specific endonucleolytic cleavage of damaged DNA. The incision required the presence of ATP indicating that functional UV-endonuclease needs ATP as a cofactor.

Carcinogens↗
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↗
PubMed1976

Reduced DNA repair in mouse satellite DNA after treatment with methylmethanesulfonate, and N-methyl-N-nitrosourea.

We have measured DNA repair in mouse satellite and main band DNA as resolved by Ag+-Cs2SO4 centrifugation in response to treatment with the alkylating agents, methyl methanesulfonate, and N-methyl-N-nitrosourea. We find that there is a statistically significant lower incorporation of 3H-Tdr into the satellite DNA as compared to the main band at varying periods after treatment with the alkylating agents. This suggests a reduced repair activity in the satellite DNA. We have measured the extent of binding of 14C-methyl methanesulfonate to the satellite, and main band DNA, and no difference in binding was observed, indicating that the reduced repair activity of satellite DNA is not due to a difference in binding of alkylating agents. We believe that the reduced incorporation of 3H-Tdr into satellite DNA may be due to its location in the condensed chromatin fraction.

Animals↗
PubMed1979

DNA repair processes protect human beings from premature solar skin damage: evidence from studies on xeroderma pigmentosum.

The repair of DNA damage by ultraviolet light is defective in the hereditary disease xeroderma pigmentosum. A deoxyribonucleotide excision-proficient form and several excision-deficient forms of xeroderma pigmentosum have been identified. Premature solar skin damage develops in all xeroderma pigmentosum patients. Some patients also have neurological abnormalities caused by premature death of nerve cells. This abnormal aging of the central nervous system and of sun-exposed skin appears to be the result of the abnormal DNA repair processes. Clinical, biological, and physicochemical studies on DNA-repair-dependent processes and on the DNA repair defects in xeroderma pigmentosum are elucidating the mechanisms by which such abnormal aging is prevented in normal human beings.

Adult↗
PubMed1979

Induction of the DNA repair enzyme uracil-DNA glycosylase in stimulated human lymphocytes.

The capacity of human cells to modulate the synthesis of DNA repair enzymes has been investigated by measuring the induction of the uracil-DNA glycosylase during lymphocyte stimulation. Treatment of peripheral lymphocytes with phytohemagglutinin increased glycosylase activity 10-fold. Glycosylase stimulation was coordinate with the activation of DNA synthesis and DNA polymerase activity. Two chromatographically distinct species of the glycosylase have been resolved; only one species is induced during phytohemagglutinin stimulation. The effect of actinomycin D and cycloheximide on glycosylase induction was determined. Treatment with either inhibitor at 96 hr after phytohemagglutinin addition (maximal induction) decreased glycosylase activity after an appreciable lag period. This suggested that induction of the uracil-DNA glycosylase requires transcription and translation although the enzyme may be quite stable once induced.

Cycloheximide↗
PubMed1977

Integration of proviral DNA in chicken cells infected with Schmidt-Ruppin Rous sarcoma virus is not enhanced by DNA repair.

The effect DNA repair might have on the integration of exogenous proviral DNA into host cell DNA was investigated by comparing the efficiency of proviral DNA integration in normal chicken embryonic fibroblasts and in chicken embryonic fibroblasts treated with UV or 4-nitroquinoline-1-oxide. The cells were treated with UV or 4-nitroquinoline-1-oxide at various time intervals ranging from 6 h before to 24 h after infection with Schmidt-Ruppin strain A of Rous sarcoma virus. The chicken embryonic fibroblasts were subsequently cultured for 18 to 21 days to ensure maximal integration and elimination of nonintegrated exogenous proviral DNA before DNA was extracted. Integration of proviral DNA into the cellular genome was quantitated by hybridization of denatured cellular DNA on filters with an excess of (3)H-labeled 35S viral RNA. The copy number of the integrated proviruses in normal cells and in infected cells was also determined from the kinetics of liquid RNA-DNA hybridization in DNA excess. Both RNA excess and DNA excess methods of hybridization indicate that two to three copies of the endogenous provirus appear to be present per haploid normal chicken cell genome and that two to three copies of the provirus of Schmidt-Ruppin strain A of Rous sarcoma virus become integrated per haploid cell genome after infection. The copy number of viral genome equivalents integrated per cell treated with UV or 4-nitroquinoline-1-oxide at different time intervals before or after infection did not differ from the copy number in untreated but infected cells. This finding supports our previous report that the integration of oncornavirus proviral DNA is restricted to specific sites in the host cell DNA and suggests a specific mechanism for integration.

4-Nitroquinoline-1-oxide↗
PubMed1979

Clinical, genetic and DNA repair studies on a consecutive series of patients with xeroderma pigmentosum.

We report clinical, genetic and biochemical findings in 13 families with the photosensitive genodermatosis, xeroderma pigmentosum. All patients had a defect in repair of DNA damage provoked by ultraviolet radiation. Eleven patients and their three affected sibs were defective in the excision repair of UVR induced DNA lesions while the other two were defective in post-replication repair. One in the former group was diagnosed prior to the development of permanent skin abnormalities and preventive measures succeeded for almost five years in maintaining a normal appearing skin. In addition, two cases were diagnosed prenatally and aborted therapeutically. Some patients' parents showed slightly reduced repair of UVR induced DNA damage. In xeroderma pigmentosum (XP), the defect in the excision of DNA lesions appears to be due to homozygosity for one of at least seven different mutations and, accordingly, XP patients can be assigned to seven so-called complementation groups, A to G. Of these, groups A, C and D are the most common. Somatic cell fusion allowed three of the families reported here to be assigned to group A, four to group C and four to group D. Fibroblasts of patients from these three groups were shown to differ not only in the degree and kinetics of their residual DNA repair but also in the kinetics with which their defect is complemented by fusion with normal or XP cells of other groups. This confirms that mutations of different genes play a role in XP and provides a basis for understanding how such genes interact to secure repair of DNA lesions in normal cells. We discuss the phenotype of XP from different complementation groups in relation to the severe neurological abnormalities which may develop and must be considered in genetic counselling. We also discuss the biochemical anomalies of XP and the cellular effects of physical and chemical agents which damage DNA. In the practical management of XP, the importance of early differential diagnosis and prompt initiation of treatment is emphasized. Lastly we review the relationship between DNA repair and skin cancer in XP.

Adolescent↗
PubMed1978

Xeroderma pigmentosum: recent studies on the DNA repair defects.

Xeroderma pigmentosum is a recessive autosomal disease of humans that is characterized by a high prevalence of skin cancers. Results of studies on cells from such patients indicate a defect in the repair of DNA damage associated with exposure to ultraviolet radiation. Since this observation was reported, a large amount of information on this disease has accumulated in the literature.

Cell Fusion↗
PubMed1979

Longevity, stability and DNA repair.

The functional capacity of a cell, tissue, organ, or organism is dependent upon its ability to maintain the stability of its unit components. The higher the differentiated state of the system, the greater the amount of stability required to maintain that state as a function of time. Stability can be achieved via either redundancy or repair. Redundancy while easily achievable in biological systems is both costly and limited by thermodynamic considerations. Repair, in its general sense, has no such limitations. Repair at the cellular and macromolecular level is multiple in its forms and varies as a function of species, tissue, and stage of the cell cycle. The repair of DNA damage is a dynamic process with many components and subcomponents, each interacting with one another in order to achieve a balance between individual stability and evolutionary diversity. Thus, between internal and external factors which damage DNA and the subsequent expression of alterations in the functional stability of DNA lie the multi-functional pathways which attempt to maintain DNA fidelity. A strong correlation between ulta-violet light induced excision or pre-replication repair, as measured by autoradiogrphy and maximum species lifespan has been reported within different strains of the same species, between related species (e.g. Mus musculus and Peromyscus leucopus), between five orders of mammals, and most recently within members of the primate family. As has been demonstrated by the authors and others, differences in excision repair between species and tissues may relate to the turning off of portions of the repair processes during embryogenesis. Regardless of why such correlations exist or the nature of their mechanisms, it is naive to either assert or deny a causal role for DNA repair in longevity assurance systems. For example, while species-related differences in DNA repair may reflect the turning off of such repair processes during fetal development this does not mean that rates of accumulation of DNA damage are not altered by such changes. Indeed, such a phenomena might well explain the rapid evolution of lifespan within the primates without a concurrent input of new genes.

Aging↗
PubMed1979

DNA damage and DNA repair in cultured human cells exposed to chromate.

DNA damage and DNA repair have been observed in cultured human skin fibroblasts exposed to potassium chromate but not to a chromic glycine complex. DNA repair synthesis (unscheduled incorporation of [3H]thymidine (TdR)) was measured in cells during or following exposure to chromate and was significant for chromate concentrations above 10(-6) M. Maximal DNA repair was observed at about 10(-4) M chromate. DNA repair capacity was found to be saturated at this concentration. Chromate was stable for at least 8 h in culture medium and produced approximately a linear increase in repair with duration of exposure. DNA damage as determined by alkaline sucrose gradient sedimentation was detected after treatment for 1.5 h with 5 . 10(-4) M chromate. Exposure to 10(-7) M chromate solution for 7 days inhibited colony formation while acute (1 h) treatment was toxic at 5 . 10(-6) M. The chromic glycine complex was toxic above 10(-3) M for a 1-week exposure but was not observably toxic after a 1-h treatment. These results indicate that chromate and not chromic compounds may be the carcinogenic form for man. The nature of the ultimate carcinogen is discussed. These findings illustrate the utility of the DNA repair technique to study the effects on human cells of inorganic carcinogens and mutagens.

Adult↗
PubMed1979

Survival and DNA repair in ultraviolet-irradiated haploid and diploid cultured frog cells.

Survival and repair of DNA following ultraviolet (254-nm) radiation have been investigated in ICR 2A, a cultured cell line from haploid embryos of the grassfrog, Rana pipiens. Survival curves from cells recovering in the dark gave mean lethal dose value (Do) in the range 1.5--1.7 Jm-2 for both haploid and diploid cell stocks. The only significant difference observed between haploids and diploids was in the extent of the shoulder at low fluence (Dq), the value for exponentially multiplying diploid cells (3.0 Jm-2) being higher than that found for haploids (1.2 Jm-2). Irradiation of cultures reversibly blocked in the G1 phase of the cell cycle gave survival-curve coefficients indistinguishable between haploids and diploids. Post-irradiation exposure to visible light restored colony-forming capacity and removed chromatographically estimated pyrimidine dimers from DNA at the same rates. After fluences killing 90% of the cells, complete restoration of survival was obtained after 60-min exposure to 500 foot-candles, indicating that in this range lethality is entirely photoreversible and therefore attributable to pyrimidine dimers in DNA. Dimer removal required illumination following ultraviolet exposure, intact cells and physiological temperature, implying that the photoreversal involved DNA photolyase activity. Excision-repair capacity was slight, since no loss of dimers could be detected chromatographically during up to 48 h incubation in the dark and since autoradiographically detected "unscheduled DNA synthesis" was limited to a 2-fold increase saturated at 10 Jm-2. These properties make ICR 2A frog cells useful to explore how DNA-repair pathways influence mutant yield.

Animals↗