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PubMedDate not supplied

Altered expression of the DNA repair protein, N-methylpurine-DNA glycosylase (MPG) in human gonads.

BACKGROUND: The multifunctional mammalian MPG is responsible for a damaged DNA base in the nucleus. The DNA repair enzyme is transported from the cytoplasm to nucleus to repair the DNA base when it is damaged. If the enzyme does not work properly, the damaged DNA may lead to carcinogenesis, cell death, aging or infertility. MATERIALS AND METHODS: This study was performed to determine mRNA expression and intracellular localization of the DNA repair protein, N-methylpurine-DNA glycosylase (MPG), in human ovary and testicular tissues, particularly in epithelial ovarian tumor and spermatogenic (maturation) arrest infertile patients, by RT-PCR and immunohistochemical staining using human MPG monoclonal antibody. RESULTS: MPG mRNA expression in epithelial ovarian tumor and spermatogenic arrest testis tissues was slightly higher than in normal ovarian and testicular tissues, respectively. The present study demonstrated new and unexpected patterns of cellular and subcellular localization of this enzyme. In a normal ovary, immunostaining for MPG was observed in the nucleus of oocyte, granulosa and stromal cells. MPG was stained mostly in the nucleus and faintly-stained in the cytoplasm of normal coelomic epithelium as well as in benign epithelial ovarian tumors. However, the MPG expression of the nucleus in malignant epithelial tumors, both serous and mucinous type, disappeared. The spermatocyte and Leydig cells in normal testis were immunostained only in the cytoplasm. The spermatocyte and Leydig cells in spermatogenic arrest testis tissues showed up both in the nucleus and cytoplasm. The subcellular localization of MPG in the tissues tested was heterogeneous, while the altered MPG expression was found in ovarian tumor and spermatogenic arrest testis. CONCLUSION: These results suggest MPG's role in human gonadal tissues and raise the possibility that the altered mRNA level and intracellular localization could be associated with ovarian tumorigenesis and male infertility.

Cytoplasm↗
PubMed1990

The regulation of DNA repair during development.

DNA repair is important in such phenomena as carcinogenesis and aging. While much is known about DNA repair in single-cell systems such as bacteria, yeast, and cultured mammalian cells, it is necessary to examine DNA repair in a developmental context in order to completely understand its processes in complex metazoa such as man. We present data to support the notion that proliferating cells from organ systems, tumors, and embryos have a greater DNA repair capacity than terminally differentiated, nonproliferating cells. Differential expression of repair genes and accessibility of chromatin to repair enzymes are considered as determinants in the developmental regulation of DNA repair.

Animals↗
PubMed1998

Nonconservative amino acid substitution variants exist at polymorphic frequency in DNA repair genes in healthy humans.

The removal or repair of DNA damage has a key role in protecting the genome of the cell from the insults of cancer-causing agents. This was originally demonstrated in individuals with the rare genetic disease xeroderma pigmentosum, the paradigm of cancer genes, and subsequently in the relationship between mismatch repair and colon cancer. Recent reports suggest that individuals with less dramatic reductions in the capacity to repair DNA damage are observed at polymorphic frequency in the population; these individuals have an increased susceptibility to breast, lung, and skin cancer. We report initial results from a study to estimate the extent of DNA sequence variation among individuals in genes encoding proteins of the DNA repair pathways. Nine different amino acid substitution variants have been identified in resequencing of the exons of three nucleotide excision repair genes (ERCC1, XPD, and XPF), a gene involved in double-strand break repair/recombination genes (XRCC3), and a gene functioning in base excision repair and the repair of radiation-induced damage (XRCCI). The frequencies for the nine different variant alleles range from 0.04 to 0.45 in a group of 12 healthy individuals; the average allele frequency is 0.17. The potential that this variation, and especially the six nonconservative amino acid substitutions occurring at residues that are identical in human and mouse, may cause reductions in DNA repair capacity or the fidelity of DNA repair is intriguing; the role of the variants as cancer risk factors or susceptibility alleles remains to be addressed.

Amino Acid Sequence↗
PubMed1994

An interaction between the mammalian DNA repair protein XRCC1 and DNA ligase III.

XRCC1, the human gene that fully corrects the Chinese hamster ovary DNA repair mutant EM9, encodes a protein involved in the rejoining of DNA single-strand breaks that arise following treatment with alkylating agents or ionizing radiation. In this study, a cDNA minigene encoding oligohistidine-tagged XRCC1 was constructed to facilitate affinity purification of the recombinant protein. This construct, designated pcD2EHX, fully corrected the EM9 phenotype of high sister chromatid exchange, indicating that the histidine tag was not detrimental to XRCC1 activity. Affinity chromatography of extract from EM9 cells transfected with pcD2EHX resulted in the copurification of histidine-tagged XRCC1 and DNA ligase III activity. Neither XRCC1 or DNA ligase III activity was purified during affinity chromatography of extract from EM9 cells transfected with pcD2EX, a cDNA minigene that encodes untagged XRCC1, or extract from wild-type AA8 or untransfected EM9 cells. The copurification of DNA ligase III activity with histidine-tagged XRCC1 suggests that the two proteins are present in the cell as a complex. Furthermore, DNA ligase III activity was present at lower levels in EM9 cells than in AA8 cells and was returned to normal levels in EM9 cells transfected with pcD2EHX or pcD2EX. These findings indicate that XRCC1 is required for normal levels of DNA ligase III activity, and they implicate a major role for this DNA ligase in DNA base excision repair in mammalian cells.

Amino Acid Sequence↗
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↗
PubMed1988

DNA repair synthesis in human fibroblasts requires DNA polymerase delta.

When UV-irradiated cultured diploid human fibroblasts were permeabilized with Brij-58 then separated from soluble material by centrifugation, conservative DNA repair synthesis could be restored by a soluble factor obtained from the supernatant of similarly treated HeLa cells. Extensive purification of this factor yielded a 10.2 S, 220,000-dalton polypeptide with the DNA polymerase and 3'- to 5'-exonuclease activities reported for DNA polymerase delta II (Crute, J. J., Wahl, A. F., and Bambara, R. A. (1986) Biochemistry 25, 26-36). Monoclonal antibody to KB cell DNA polymerase alpha, while binding to HeLa DNA polymerase alpha, did not bind to the HeLa DNA polymerase delta. Moreover, at micromolar concentrations N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate (BuPdGTP) and 2-(p-n-butylanilino)-2'-deoxyadenosine 5'-triphosphate (BuAdATP) were potent inhibitors of DNA polymerase alpha, but did not inhibit the DNA polymerase delta. Neither purified DNA polymerase alpha nor beta could promote repair DNA synthesis in the permeabilized cells. Furthermore, under conditions which inhibited purified DNA polymerase alpha by greater than 90%, neither monoclonal antibodies to DNA polymerase alpha, BuPdGTP, nor BuAdATP was able to inhibit significantly the DNA repair synthesis mediated by the DNA polymerase delta. Thus, it appears that a major portion of DNA repair synthesis induced by UV irradiation might be catalyzed by DNA polymerase delta. When xeroderma pigmentosum human diploid fibroblasts were utilized, DNA repair synthesis dependent upon ultraviolet light could be restored by addition of both T4 endonuclease V and DNA polymerase delta, but not by addition of either one alone. This result suggests that cytosol-depleted permeabilized DNA repair-defective human fibroblasts and HeLa DNA polymerase delta might be exploited to provide a functional assay for purifying active DNA repair factors from DNA repair-proficient cells without a preknowledge of their function.

DNA Polymerase II↗
PubMedDate not supplied

Interactions of viruses with the cellular DNA repair machinery.

Mammalian cells are equipped with complex machinery to monitor and repair damaged DNA. In addition to responding to breaks in cellular DNA, recent studies have revealed that the DNA repair machinery also recognizes viral genetic material. We review some examples that highlight the different strategies that viruses have developed to interact with the host DNA repair apparatus. While adenovirus (Ad) inactivates the host machinery to prevent signaling and concatemerization of the viral genome, other viruses may utilize DNA repair to their own advantage. Viral interactions with the repair machinery can also have detrimental consequences for the host cells and their ability to maintain the integrity of the host genome. Exploring the interactions between viruses and the host DNA repair machinery has revealed novel host responses to virus infections and has provided new tools to study the DNA damage response.

Adenoviridae↗
PubMed1983

Isolation and characterization of monoclonal antibodies directed against the DNA repair enzyme uracil DNA glycosylase from human placenta.

A series of monoclonal antibodies has been prepared against the base excision repair enzyme uracil DNA glycosylase isolated from human placenta. Spleen cells from BALB/c mice immunized with purified human placental uracil DNA glycosylase were fused with either P3X63 Ag8.653 or SP2/0 myeloma cells. Hybridomas producing antibodies directed against the placental glycosylase were identified in an enzyme-linked immunosorbent assay. Each positive hybridoma was cloned twice by limit dilution and tested for anti-glycosylase activity in an enzyme immunoprecipitation assay. Each of the four clones examined in detail precipitated enzyme activity in an immunoprecipitation reaction only in the presence of rabbit anti-mouse IgG as a second antibody. No anti-uracil DNA glycosylase activity was observed in a spontaneous hybridoma used as a control. Each monoclonal antibody immunoprecipitated uracil DNA glycosylases isolated from several human tissues. Partial crossreactivity was observed with rat liver glycosylase and with a hamster enzyme. In contrast, no crossreactivity was observed with yeast or Escherichia coli glycosylase. Glycerol gradient sedimentation analysis demonstrated that one of the antibodies bound to the glycosylase at a site that did not diminish its catalytic activity. A second monoclonal antibody bound at a determinant that affected catalytic activity. Analysis of antibody-glycosylase interactions suggests that human cells contain antigenically distinct glycosylase species that may be encoded by individual uracil DNA glycosylase genes. The potential use of these monoclonal antibodies in studies examining the regulation of glycosylase isoenzymes during cell proliferation in normal human cells and in cells from cancer-prone individuals is considered.

Animals↗
PubMed2003

Visualization of DNA-induced conformational changes in the DNA repair kinase DNA-PKcs.

The catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) is essential for the repair of double-stranded DNA breaks (DSBs) in non- homologous end joining (NHEJ) and during V(D)J recombination. DNA-PKcs binds single- and double-stranded DNA in vitro, and in vivo the Ku heterodimer probably helps recruit it to DSBs with high affinity. Once loaded onto DNA, DNA-PKcs acts as a scaffold for other repair factors to generate a multiprotein complex that brings the two DNA ends together. Human DNA-PKcs has been analysed by electron microscopy in the absence and presence of double-stranded DNA, and the three-dimensional reconstruction of DNA-bound DNA-PKcs displays large conformational changes when compared with the unbound protein. DNA-PKcs seems to use a palm-like domain to clip onto the DNA, and this new conformation correlates with the activation of the kinase. We suggest that the observed domain movements might help the binding and maintenance of DNA-PKcs' interaction with DNA at the sites of damage, and that these conformational changes activate the kinase.

Ataxia Telangiectasia Mutated Proteins↗
PubMed1998

Variation in DNA repair is a factor in cancer susceptibility: a paradigm for the promises and perils of individual and population risk estimation?

The repair of DNA damage protects the genome of the cell from the insults of cancer causing agents. This was originally demonstrated in individuals with the rare genetic disease, xeroderma pigmentosum, the prototype of cancer genes, and subsequently in the relationship of mismatch repair to colon cancer. Recent studies suggest that individuals with less dramatic reductions in the capacity to repair DNA damage are observed at polymorphic frequency and these individuals have an increased susceptibility to several types of cancer. Screening of individuals for DNA sequence variation in the exons of 9 DNA repair genes has resulted in identification of 15 different polymorphic amino acid substitution variants. Although the studies to relate these variants to reduced DNA repair capacity and cancer status have not been completed, the available information is sufficient to suggest that DNA repair genes should be incorporated into molecular epidemiology and cancer susceptibility studies. The availability of molecular epidemiology data presents exciting opportunities for refinement of risk estimation models and identification of individuals at increased risk of disease, with resultant opportunities for effective surveillance and early intervention and treatment. The opportunities to acquire susceptibility data are associated with possible perils for establishment of regulations for permissible exposures to carcinogenic agents and also stigmatization of 'at risk' individuals that may result in decreased access to employment opportunities and health care.

Animals↗
PubMed1975

DNA repair in DNA-polymerase-deficient mutants of Escherichia coli.

Escherichia coli mutants deficient in DNA polymerase I, in DNA polymerases I and II, or in DNA polymerase III can efficiently and completely execute excision-repair and postreplication repair of the UV-damaged DNA at 30 degrees C and 43 degrees C when assayed by alkaline sucrose gradients. Repair by Pol I- and Pol I-, Pol II- cells is inhibited by 1-beta-D-arabinofuranosylcytosine (araC) at 43 degrees C but not at 30 degrees C, whereas that by Pol III- cells is insensitive to araC at any temperature. Thus, either Pol I or Pol III is required for complete and efficient repair, and in their absence Pol II mediates a limited, incomplete dark repair of UV-damaged DNA.

Cytarabine↗
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↗
PubMed1992

Redox activation of Fos-Jun DNA binding activity is mediated by a DNA repair enzyme.

The DNA binding activity of Fos and Jun is regulated in vitro by a post-translational mechanism involving reduction-oxidation. Redox regulation occurs through a conserved cysteine residue located in the DNA binding domain of Fos and Jun. Reduction of this residue by chemical reducing agents or by a ubiquitous nuclear redox factor (Ref-1) recently purified from Hela cells, stimulates AP-1 DNA binding activity in vitro, whereas oxidation or chemical modification of the cysteine has an inhibitory effect on DNA binding activity. Here we demonstrate that the protein product of the ref-1 gene stimulates the DNA binding activity of Fos-Jun heterodimers, Jun-Jun homodimers and Hela cell AP-1 proteins as well as that of several other transcription factors including NF-kappa B, Myb and members of the ATF/CREB family. Furthermore, immunodepletion analysis indicates that Ref-1 is the major AP-1 redox activity in Hela nuclear extracts. Interestingly, Ref-1 is a bifunctional protein; it also possesses an apurinic/apyrimidinic (AP) endonuclease DNA repair activity. However, the redox and DNA repair activities of Ref-1 can, in part, be distinguished biochemically. This study suggests a novel link between transcription factor regulation, oxidative signalling and DNA repair processes in higher eukaryotes.

Amino Acid Sequence↗
PubMedDate not supplied

Ocular manifestations in the inherited DNA repair disorders.

Deoxyribonucleic acid (DNA) repair is a fundamental process designed to keep the integrity of genomic DNA that is continuously challenged by intrinsic or environmental induced alterations. Numerous genes involved in DNA repair have been cloned and are involved in different DNA repair pathways: base excision repair, nucleotide excision repair, mismatch repair, DNA recombination. Inherited conditions due to mutations in DNA repair genes include mainly: xeroderma pigmentosum, Cockayne syndrome, Trichothiodystrophy, Bloom syndrome, Rothmund-Thomson syndrome, and Werner syndrome. Minor to major ocular manifestations occur in these syndromes. For example, eyelid skin cancers in xeroderma pigmentosum and retinal dystrophy in Cockayne syndrome are major features of these syndromes. This review focuses on the DNA repair pathways, the general and ocular features of the related syndromes, the laboratory tests useful for diagnosis, and the general processes implied with DNA repair (ultraviolet sensitivity, carcinogenesis, apoptosis, oxydative stress, and premature aging).

Base Pair Mismatch↗
PubMed2004

[Progress of enzyme in mitochondrial DNA repair system].

Mitochondrial DNA (mtDNA) encodes subunits of the mitochondrial electron transport system and the rRNAs and tRNAs required for constructing the mitochondrial translational machinery. Each subunit encoded by mtDNA is essential for normal oxidative phosphorylation. Thus, integrity of the mtDNA is crucial for the survival of organisms. It has long been held that there is no DNA repair in mitochondria. But in recent years,a number of repair factors have been found in mitochondrial extracts, suggesting the presence of DNA repair in mitochondria. This review summarized recent progress of enzyme in mitochondrial DNA repair processes.

Animals↗
PubMed1998

H-NS regulates DNA repair in Shigella.

We report a new role for H-NS in Shigella spp.: suppression of repair of DNA damage after UV irradiation. H-NS-mediated suppression of virulence gene expression is thermoregulated in Shigella, being functional at 30 degrees C and nonfunctional at 37 to 40 degrees C. We find that H-NS-mediated suppression of DNA repair after UV irradiation is also thermoregulated. Thus, Shigella flexneri M90T, incubated at 37 or 40 degrees C postirradiation, shows up to 30-fold higher survival than when incubated at 30 degrees C postirradiation. The hns mutants BS189 and BS208, both of which lack functional H-NS, show a high rate of survival (no repression) whether incubated at 30 or 40 degrees C postirradiation. Suppression of DNA repair by H-NS is not mediated through genes on the invasion plasmid of S. flexneri M90T, since BS176, cured of plasmid, behaves identically to the parental M90T. Thus, in Shigella the nonfunctionality of H-NS permits enhanced DNA repair at temperatures encountered in the human host. However, pathogenic Escherichia coli strains (enteroinvasive and enterohemorrhagic E. coli) show low survival whether incubated at 30 or 40 degrees C postirradiation. E. coli K-12 shows markedly different behavior; high survival postirradiation at both 30 and 40 degrees C. These K-12 strains were originally selected from E. coli organisms subjected to both UV and X irradiation. Therefore, our data suggest that repair processes, extensively described for laboratory strains of E. coli, require experimental verification in pathogenic strains which were not adapted to irradiation.

Bacterial Proteins↗
PubMed1998

Mutations in hamster single-strand break repair gene XRCC1 causing defective DNA repair.

The molecular basis for the DNA repair dysfunction observed in mutant Chinese hamster ovary cell lines of X-ray repair cross complementing group 1 (XRCC1) is unknown and the exact role of the XRCC1 protein remains unclear. To help clarify the role of the XRCC1 gene we analyzed four mutant cell lines of this complementation group and a revertant cell line for XRCC1 protein content and for sequence alterations in the XRCC1 coding region. Immunoblot analysis of cellular extracts indicated that each of four mutant lines was lacking XRCC1 protein, whereas the repair-proficient revertant line derived from one of these mutants contained a normal level of XRCC1. Although each of these cell lines expressed XRCC1 mRNA, we found in all cases a distinct point mutation resulting in crucial alterations in the encoded XRCC1 protein sequence of 633 amino acids. Two of the mutations cause non-conservative amino acid changes, Glu102-->Lys and Cys390-->Tyr, at positions that are invariant among hamster, mouse and human XRCC1 sequences and are located in putative functional domains. A third debilitating mutation disrupts RNA splicing, generating multiple transcripts of different length that contain deletions spanning a region of >100 amino acids in the midsection of the XRCC1 coding sequence. A fourth mutation results in a termination codon that shortens the open reading frame to 220 amino acids, however, in the revertant cell line a further mutation in the same codon, Stop221-->Leu, permits translation of a full-length functional variant protein. These mutational data indicate the importance of the putative functional regions in XRCC1, such as the BRCA1 C-terminal (BRCT) domain found in common with BRCA1 and other DNA repair and cell cycle checkpoint proteins, and also regions necessary for interaction with DNA polymerase beta and DNA ligase III.

Alternative Splicing↗