Repair Enzyme Uracil DNA Glycosylase Research Articles

Uracil-DNA glycosylase activity in human acute leukemia

The expression of the DNA excision repair enzyme uracil-DNA glycosylase was investigated in bone marrow and peripheral samples from seven patients with acute lymphoblastic leukemia (ALL), from 17 patients with acute non-lymphocytic leukemia (ANLL), and from one patient with chronic granulocytic leukemia (CGL) in blast crisis. In addition, uracil-DNA glycosylase activities were determined in nine human leukemia/lymphoma cell lines. There was a clear correlation between the percentage of blast cells and the enzyme activity, when mononuclear cell fractions from patient samples were analysed. The following uracil-DNA glycosylase activities were recorded (mean ± S.D., number of samples): ALL = 45.6 ± 14.8 U/mg of protein, N = 10; ANLL = 41.1 ± 13.8 U/mg of protein, N = 22; CGL (blast crisis) = 44.7 U/mg of protein. The uracil-DNA glycosylase activity in nine human leukemia/lymphoma cell lines ranged from 35.2 to 66.0 U/mg of protein, and no striking differences were observed between the T-ALL, B-ALL, null cell ALL or myeloid lines. Similarly, the various biological features, such as the common ALL surface antigen, the terminal deoxynucleotidyl transferase enzyme, the subtype of leukemia, chromosomal aberrations, or previous chemotherapy, did not apparently affect the expression of uracil-DNA glycosylase. We propose that the integrity of the genetic information is well protected by uracil-DNA glycosylase in different forms of leukemia, including cases with a low proportion of S-phase blasts, as assessed by flow cytometry in the present work. When compared to the activities in benign hematopoietic progenitor cells, studied previously in this laboratory, no big differences between the benign and malignant hematopoiesis were demonstrated. Hence, it is unlikely that selectivity of chemotherapy towards malignant vs benign hematopoietic growth could be based on the enzyme uracil-DNA glycosylase.

Hematopoietic and gastric uracil-DNA glycosylase activity in megaloblastic anemia and in atrophic gastritis with special reference to pernicious anemia.

The activity of the DNA excision repair enzyme uracil-DNA glycosylase was measured in peripheral blood mononuclear cells and in bone marrow aspiration samples obtained from patients with pernicious anemia (PA) or other types of megaloblastic anemia (one case of tapeworm anemia and three cases of myelodysplastic syndromes). In addition, the expression of uracil-DNA glycosylase was investigated in biopsies from the antrum and body of the stomach obtained from nine PA patients, from five patients having atrophic gastritis (AG) not associated with PA, and from six control patients having transient upper abdominal complaints without AG. Our results revealed that there was a considerable interindividual variation in gastric uracil-DNA glycosylase activity. No clear correlation between the enzyme level and the level of gastric atrophy was noted, although AG is generally regarded as a risk factor of gastric cancer. Furthermore, uracil-DNA glycosylase activities in peripheral blood mononuclear cells and in bone marrow cells in PA and in myelodysplastic syndromes were similar to the activities observed previously in non-hematological patients and healthy persons. Transient uracil incorporation into DNA may have a role in the cellular abnormalities associated with megaloblastic hematopoiesis. The present findings demonstrated that the enzymatic activity required for rapid removal of uracil from DNA is also expressed in the megaloblastic state.

Physical association of the human base-excision repair enzyme uracil DNA glycosylase with the 70,000-dalton catalytic subunit of DNA polymerase alpha.

A monoclonal antibody prepared against a partially purified human uracil DNA glycosylase was found, on further purification of the enzyme, to be inactive against the glycosylase. However, immunoreactivity was observed in other protein fractions that contained DNA polymerase activity. The immunoreactive protein was purified to homogeneity and identified as a catalytic subunit of DNA polymerase alpha by molecular mass, by aphidicolin sensitivity, and by recognition by a monoclonal antibody against human KB cell DNA polymerase alpha. Our monoclonal antibody had no effect on homogeneous human uracil DNA glycosylase activity but severely inhibited the activity of the homogeneous human DNA polymerase alpha catalytic subunit. The suspicion that the two proteins were physically associated was confirmed by finding that, on mixing the DNA polymerase alpha subunit with the glycosylase, the latter was strongly inhibited by our monoclonal antibody. These results demonstrate that this monoclonal antibody recognizes not only the DNA polymerase alpha subunit but also the uracil DNA glycosylase when it is physically attached to the polymerase subunit. These results contribute to the definition of relationships between those proteins that may comprise the human base-excision repair multienzyme complex.

Regulation of DNA repair in serum-stimulated xeroderma pigmentosum cells.

The regulation of DNA repair during serum stimulation of quiescent cells was examined in normal human cells, in fibroblasts from three xeroderma pigmentosum complementation groups (A, C, and D), in xeroderma pigmentosum variant cells, and in ataxia telangiectasia cells. The regulation of nucleotide excision repair was examined by exposing cells to ultraviolet irradiation at discrete intervals after cell stimulation. Similarly, base excision repair was quantitated after exposure to methylmethane sulfonate. WI-38 normal human diploid fibroblasts, xeroderma pigmentosum variant cells, as well as ataxia telangiectasia cells enhanced their capacity for both nucleotide excision repair and for base excision repair prior to their enhancement of DNA synthesis. Further, in each cell strain, the base excision repair enzyme uracil DNA glycosylase was increased prior to the induction of DNA polymerase using the identical cells to quantitate each activity. In contrast, each of the three xeroderma complementation groups that were examined failed to increase their capacity for nucleotide excision repair above basal levels at any interval examined. This result was observed using either unscheduled DNA synthesis in the presence of 10 mM hydroxyurea or using repair replication in the absence of hydroxyurea to quantitate DNA repair. However, each of the three complementation groups normally regulated the enhancement of base excision repair after methylmethane sulfonate exposure and each induced the uracil DNA glycosylase prior to DNA synthesis. These results suggest that there may be a relationship between the sensitivity of xeroderma pigmentosum cells from each complementation group to specific DNA damaging agents and their inability to regulate nucleotide excision repair during cell stimulation.

The cloning and overproduction of Escherichia coli uracil-DNA glycosylase

Escherichia coli cells containing elevated levels of the DNA repair enzyme uracil-DNA glycosylase (the ung gene product) have been constructed by in vitro recombination methods. First, λ nadB transducing phages were isolated from two E. coli DNA libraries by selection of nicotinate-independent lysogens. λ nadB phage from one of the libraries were also ung + and carried the ung-nadB genes on an 8.3 kb HindIII restriction fragment. The ung and nadB genes were subcloned into plasmids and a restriction map of the ung region of the E. coli chromosome was constructed. The uracil glycosylase gene was localized to a 1.4-kb restriction fragment by subcloning the gene into pBR322. Uracil glycosylase was overproduced (relative to the specific activity of wild type cells) by about two-fold in λ ung lysogens and by 15- to 20-fold in cells containing pBR322 ung derivatives. When the ung gene and its promoter were placed downstream from the bacteriophage λp L promoter in the plasmid pKC30, uracil glycosylase production was heat-induced to more than 100-fold above the levels of a wild-type cell. By relating the insertion orientation of the λ ung gene in the plasmid pKC30 to its orientation in λ ung-nadB transducing phages, the transcription direction of the ung gene on the E. coli linkage map was found to be clockwise.

Altered temporal expression of DNA repair in hypermutable Bloom's syndrome cells.

The temporal regulation of DNA repair during synchronous cell proliferation was examined in normal human skin fibroblasts and in Bloom's syndrome skin fibroblasts. Normal human cells regulated DNA repair in a defined temporal sequence prior to the induction of DNA replication. Nucleotide-excision repair was stimulated prior to the induction of base-excision repair, which itself was increased prior to the induction of DNA replication. This temporal sequence was observed (i) by quantitation of the induction of the base-excision repair enzyme uracil DNA glycosylase during cell proliferation in the absence of cellular insult and (ii) by quantitation of nucleotide-excision repair after UV irradiation or base-excision repair after exposure to methylmethane sulfonate. In contrast, Bloom's syndrome cells were characterized by specific alterations in this temporal sequence of gene regulation, such that DNA repair was not enhanced prior to the induction of DNA replication. Nucleotide-excision repair, base-excision repair, and the uracil DNA glycosylase were induced in a temporal sequence identical to that observed for DNA polymerase and for DNA replication. The inability of Bloom's syndrome cells to enhance DNA repair prior to DNA replication suggests that miscoding lesions remain in DNA and are replicated during cell proliferation.

A simplified assay for O6-methylguanine-DNA methyltransferase activity and its application to human neoplastic and non-neoplastic tissues

A rapid assay of O6-MeG-DNA methyltransferase activity is described. Following incubation of cell extracts with O6-[3H]MeG-containing DNA, remaining radioactive DNA was hydrolyzed in trichloroacetic acid and separated from methylated radioactive protein by filtration or centrifugation. Transfer of radioactive methyl from DNA to protein was proportional to the amount of protein added, and was not linear with time. More than 90% of the radioactivity precipitated after acid hydrolyses was in S-methyl cysteine residues. The method was used to measure O6-MeG-DNA methyltransferase activity in extracts of 24 neoplastic tissues from human organs. Although five tumor tissues had 28-84% lower activity of O6-MeG-DNA methyltransferase than the corresponding normal tissue from the same patient, higher or similar levels of activity were found more frequently. Thus, a lack of O6-MeG-DNA methyltransferase activity in human tumours appears not to be a frequent event. The DNA repair enzyme uracil-DNA glycosylase was also measured in the same extracts. Most frequently the level of uracil-DNA glycosylase activity was essentially similar in tumors and normal tissues but significantly higher or lower levels were also observed.

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.