Biomedical subjects

G Slupphaug

Publications and source records attributed to G Slupphaug.

27 records · Page 2Linked to original sources

PubMed1995

Crystal structure and mutational analysis of human uracil-DNA glycosylase: structural basis for specificity and catalysis.

Crystal structures of the DNA repair enzyme human uracil-DNA glycosylase (UDG), combined with mutational analysis, reveal the structural basis for the specificity of the enzyme. Within the classic alpha/beta fold of UDG, sequence-conserved residues form a positively charged, active-site groove the width of duplex DNA, at the C-terminal edge of the central four-stranded parallel beta sheet. In the UDG-6-aminouracil complex, uracil binds at the base of the groove within a rigid preformed pocket that confers selectivity for uracil over other bases by shape complementary and by main chain and Asn-204 side chain hydrogen bonds. Main chain nitrogen atoms are positioned to stabilize the oxyanion intermediate generated by His-268 acting via nucleophilic attack or general base mechanisms. Specific binding of uracil flipped out from a DNA duplex provides a structural mechanism for damaged base recognition.

Amino Acid Sequence↗

PubMed1995

Properties of a recombinant human uracil-DNA glycosylase from the UNG gene and evidence that UNG encodes the major uracil-DNA glycosylase.

We have expressed a human recombinant uracil-DNA glycosylase (UNG delta 84) closely resembling the mature form of the human enzyme (UNG, from the UNG gene) in Escherichia coli and purified the protein to apparent homogeneity. This form, which lacks the first seven nonconserved amino acids at the amino terminus, has properties similar to a 50% homogeneous UDG purified from human placenta except for a lower salt optimum and a slightly lower specific activity. The recombinant enzyme removed U from ssDNA approximately 3-fold more rapidly than from dsDNA. In the presence of 10 mM NaCl, Km values were 0.45 and 1.6 microM with ssDNA and dsDNA, respectively, but Km values increased significantly with higher NaCl concentrations. The pH optimum for UNG delta 84 was 7.7-8.0; the activation energy, 50.6 kJ/mol; and the pI between 10.4 and 10.8. The enzyme displays a striking sequence specificity in removal of U from UA base pairs in M13 dsDNA. The sequence specificity for removal of U from UG mismatches (simulating the situation after deamination of C) was essentially similar to removal from UA matches when examined in oligonucleotides. However, removal of U from UG mismatches was in general slightly faster, and in some cases significantly faster, than removal from UA base pairs. Immunofluorescence studies using polyclonal antibodies against UNG delta 84 demonstrated that the major fraction of UNG was located in the nucleus. Furthermore, > 98% of the total uracil-DNA glycosylase activity from HeLa cell extracts was inhibited by the antibodies, indicating that the UNG protein represents the major uracil-DNA glycosylase in the cells.

Base Sequence↗

PubMed1995

Cell cycle regulation and subcellular localization of the major human uracil-DNA glycosylase.

The subcellular localization of the human DNA-repair enzyme uracil-DNA glycosylase from the UNG gene has been studied using flow cytometry and laser scanning confocal microscopy of freely cycling HeLa S3 cells. A two-parameter flow cytometric analysis using propidium iodide and UNG-specific antibodies demonstrated that total cellular UNG increased during the G1-phase and was approximately doubled in early S-phase compared to early G1. The UNG level was stable during the S-phase and increased further during G2, reaching a 2.8-fold level compared to early G1. This factor included differences in cell size and staining variabilities. These findings were confirmed using two-parameter confocal analysis of UNG/DNA and UNG/mitochondria at different stages of the cell cycle. Although the major fraction of UNG was associated with nuclei, we also observed distinctive staining associated with mitochondria and a more diffuse staining probably reflecting UNG in the cytosol. Furthermore, very little UNG staining was observed in nucleoli. The UNG level in different cell compartments varied at different stages of the cell cycle, and this variation was most pronounced in the nuclei. These results demonstrate that the gene product from the UNG gene is located within three subcellular compartments and that the distribution between these compartments varies during the cell cycle.

Cell Cycle↗

PubMed1993

Nuclear and mitochondrial forms of human uracil-DNA glycosylase are encoded by the same gene.

Recent cloning of a cDNA (UNG15) encoding human uracil-DNA glycosylase (UDG), indicated that the gene product of M(r) = 33,800 contains an N-terminal sequence of 77 amino acids not present in the presumed mature form of M(r) = 25,800. This led to the hypothesis that the N-terminal sequence might be involved in intracellular targeting. To examine this hypothesis, we analysed UDG from nuclei, mitochondria and cytosol by western blotting and high resolution gel filtration. An antibody that recognises a sequence in the mature form of the UNG protein detected all three forms, indicating that they are products of the same gene. The nuclear and mitochondrial form had an apparent M(r) = 27,500 and the cytosolic form an apparent M(r) = 38,000 by western blotting. Gel filtration gave essentially similar estimates. An antibody with specificity towards the presequence recognised the cytosolic form of M(r) = 38,000 only, indicating that the difference in size is due to the presequence. Immunofluorescence studies of HeLa cells clearly demonstrated that the major part of the UDG activity was localised in the nuclei. Transfection experiments with plasmids carrying full-length UNG15 cDNA or a truncated form of UNG15 encoding the presumed mature UNG protein demonstrated that the UNG presequence mediated sorting to the mitochondria, whereas UNG lacking the presequence was translocated to the nuclei. We conclude that the same gene encodes nuclear and mitochondrial uracil-DNA glycosylase and that the signals for mitochondrial translocation resides in the presequence, whereas signals for nuclear import are within the mature protein.

Amino Acid Sequence↗

PubMed1993

Low incorporation of dUMP by some thermostable DNA polymerases may limit their use in PCR amplifications.

Incorporation of dUMP instead of dTMP is frequently used to control carryover contamination during PCR amplifications. We have tested four thermostable DNA polymerases for their ability to utilize dUTP as a substrate in PCR. Amplification of products in the presence of dUTP instead of dTTP was good with Thermus aquaticus DNA polymerase but highly inefficient with three other thermostable DNA polymerases. The latter was due to: (a) lower incorporation of dUMP relative to dTMP, (b) increased proofreading toward dUMP in DNA, (c) relative termination at dUMP residues as verified by sequencing reactions in the presence of dUTP, (d) thermostable dUTPase activity in the commercial enzyme preparation. The last point only applies to Pyrococcus furiosus DNA polymerase. This study demonstrates that various thermostable DNA polymerases utilize dTTP and dUTP with highly different efficiencies and thus the choice of DNA polymerase may be critical for amplification of DNA.

Bacteria↗

PubMed1993

Consensus sequences for good and poor removal of uracil from double stranded DNA by uracil-DNA glycosylase.

We have purified uracil DNA-glycosylase (UDG) from calf thymus 32,000-fold and studied its biochemical properties, including sequence specificity. The enzyme is apparently closely related to human UDG, since it was recognised by a polyclonal antibody directed towards human UDG. SDS-PAGE and western analysis indicate an apparent M(r) = 27,500. Bovine UDG has a 1.7-fold preference for single stranded over double stranded DNA as a substrate. Sequence specificity for uracil removal from dsDNA was examined for bovine and Escherichia coli UDG, using DNA containing less than one dUMP residue per 100 nucleotides and synthetic oligonucleotides containing one dUMP residue. Comparative studies involving about 40 uracil sites indicated similar specificities for both UDGs. We found more than a 10-fold difference in rates of uracil removal between different sequences. 5'-G/CUT-3' and 5'-G/CUG/C-3' were consensus sequences for poor repair whereas 5'-A/TUAA/T-3' was a consensus for good repair. Sequence specificity was verified in double stranded oligonucleotides, but not in single stranded ones, suggesting that the structure of the double stranded DNA helix has influence on sequence specificity. Rate of uracil removal appeared to be slightly faster from U:A base pairs as compared to U:G mis-matches. The results indicate that sequence specific repair may be a determinant to be considered in mutagenesis.

Animals↗

PubMed1992

Expression of O6-methylguanine--DNA methyltransferase and uracil--DNA glycosylase in human placentae from smokers and non-smokers.

DNA repair capacity is likely to be a critical factor in mutagenesis and carcinogenesis, as well as for the response to some cytostatics. We have studied inter- and intra-individual variation in the activities of O6-methylguanine--DNA methyltransferase (O6-MT) and uracil--DNA glycosylase (UDG) in 35 placentae from smokers and non-smokers. The maximum interindividual variation in the activities of O6-MT and UDG were 8.3- and 7.7-fold, respectively. The corresponding intraindividual variations were 2.7- and 3.3-fold. Generally, a high level of O6-MT activity was accompanied by a high O6-MT mRNA level, but no such correlation was seen for UDG. These results were not due to degradation of the enzymes or mRNAs after delivery. No correlation between the activities of O6-MT and UDG was observed, indicating that they are differentially regulated. A 1.4-fold (P < or = 0.05) higher activity of O6-MT was observed in smokers as compared to non-smokers, indicating a small, but statistically significant difference. No significant difference was observed for UDG. Our results demonstrate that DNA repair capacities vary largely between different individuals, and that environmental factors may modulate the expression of DNA repair enzymes.

DNA Glycosylases↗

PubMed1991

Cell cycle regulation and in vitro hybrid arrest analysis of the major human uracil-DNA glycosylase.

Uracil-DNA glycosylase (UDG) is the first enzyme in the excision repair pathway for removal of uracil in DNA. In vitro transcription/translation of a cloned human cDNA encoding UDG resulted in easily measurable UDG activity. The apparent size of the primary translation product was 34 kD. Two lines of evidence indicated that this cDNA encodes the major nuclear UDG. First, in vitro translation of human fibroblast mRNA isolated from S-phase cells resulted in measurable UDG activity and this UDG translation was specifically inhibited 90% by an anti-sense UDG mRNA transcript. Secondly, cell cycle analysis revealed an 8-12 fold increase in transcript level late in the G1-phase preceding a 2-3 fold increase in total UDG activity in the S-phase. UDG degradation was found to be very slow (T1/2 approximately 30h), therefore, the rate of UDG synthesis could be derived from the rate of UDG accumulation, and was found to correlate temporarily and quantitatively with the transcript level. Inhibitor studies showed that RNA and protein synthesis was required for induction of UDG. However, specific inhibition of DNA replication with aphidicolin indicated that entrance of fibroblasts into the S-phase was not required for UDG accumulation.

Aphidicolin↗

PubMed1990

Glucocorticoids inhibit the production of IL6 from monocytes, endothelial cells and fibroblasts.

We have examined the effect of dexamethasone (DM) and cortisol on the production of interleukin (IL)6 from the murine macrophage cell line RAW264.9, human monocytes, human endothelial cells and the human fibroblast cell line FS4. In RAW264.9 cells DM in the concentration range 10(-9) M to 10(-6) M inhibited the lipopolysaccharide (LPS)-induced production of IL6 by 10% to 90%. Cortisol had a similar effect, but was about 25 times less potent than DM. Also, when 10(-6) M of DM was added to the cultures after addition of LPS, it completely inhibited the residual 24-h production of IL6. Corresponding to the effect on IL6 production, DM (10(-6) M) reduced the mRNA levels for IL6 in the RAW264.9 cells. The glucocorticoid analogue RU 486 competes with DM and cortisol for the glucocorticoid receptor and reversed the inhibitory effect of DM, demonstrating that DM exerts its effect via the glucocorticoid receptor. DM also had an inhibitory effect on LPS-stimulated IL6 production in freshly isolated human monocytes, and on IL 1-stimulated IL6 production in human endothelial cells and FS4 fibroblasts. These results demonstrate that DM via a receptor-mediated mechanism inhibits IL6 production at the transcriptional level, and this may contribute to the anti-inflammatory and immunosuppressive effect of glucocorticoids.

Animals↗