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S H Wilson

Publications and source records attributed to S H Wilson.

At least 55 records · Page 3Linked to original sources
PubMed2000

8-oxodGTP incorporation by DNA polymerase beta is modified by active-site residue Asn279.

To understand how the active site of a DNA polymerase might modulate the coding of 8-oxo-7,8-dihydrodeoxyguanine (8-oxodG), we performed steady-state kinetic analyses using wild-type DNA polymerase beta (pol beta) and two active-site mutants. We compared the coding of these polymerases by calculating the ratio of efficiencies for incorporation of dATP and dCTP opposite 8-oxodG and for incorporation of 8-oxodGTP opposite dA and dC. For wild-type pol beta, there is a 2:1 preference for incorporation of dCTP over dATP opposite 8-oxodG using a 5'-phosphorylated 4-base gap substrate. Mutation of either Asn279 or Arg283 to alanine has almost no effect on the ratio. 8-OxodGTP is preferentially incorporated opposite a template dA (24:1) by wild-type pol beta; mutation of Asn279 to alanine results dramatic change whereby there is preferential incorporation of 8-oxodGTP opposite dC (14:1). This suggests that interactions of 8-oxodGTP with Asn279 in the polymerase active site may alter the conformation of 8-oxodGTP and therefore alter its misincorporation.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2000

Kinetic analysis of Sp1-mediated transcriptional activation of a TATA-containing promoter.

Using a HeLa cell nuclear extract (NE)-based in vitro runoff transcription system, we have examined the effect of Sp1 on the activation of a TATA-containing chimeric DNA polymerase beta (pAS8) promoter. The results demonstrated that the TATA element-dependent basal activity of the pAS8 promoter was stimulated 4-fold by supplementation of a Sp1-depleted HeLa cell nuclear extract (NEd) with purified human Sp1, indicating that pAS8 promoter activity is dependent upon Sp1. A detailed kinetic analysis based on a three-step kinetic model of transcription initiation showed that Sp1 stimulates the activity of the pAS8 promoter by increasing the amount of closed preinitiation complex (RP(c)) assembly as well as by enhancing the rate of promoter clearance (k(3)). There was no significant effect of Sp1 on the apparent rate of open complex (RP(o)) formation (k(2)) of the pAS8 promoter. These studies define more precisely the kinetic mechanisms by which Sp1 may regulate the rate of transcript formation of a TATA-containing promoter.

Base Sequence↗
PubMed2000

Protection against methylation-induced cytotoxicity by DNA polymerase beta-dependent long patch base excision repair.

Using a plasmid-based uracil-containing DNA substrate, we found that the long patch base excision repair (BER) activity of a wild-type mouse fibroblast extract was partially inhibited by an antibody to DNA polymerase beta (beta-pol). This suggests that beta-pol participates in long patch BER, in addition to single-nucleotide BER. In single-nucleotide BER, the deoxyribose phosphate (dRP) in the abasic site is removed by the lyase activity of beta-pol. Methoxyamine (MX) can react with the aldehyde of an abasic site, making it refractory to the beta-elimination step of the dRP lyase mechanism, thus blocking single-nucleotide BER. MX exposure sensitizes wild-type, but not beta-pol null mouse embryonic fibroblasts, to the cytotoxic effects of methyl methanesulfonate (MMS) and methylnitrosourea. Expression of beta-pol in the null cells restores the ability of MX to modulate sensitivity to MMS. The beta-pol null cells are known to be hypersensitive to MMS and methylnitrosourea, and in the presence of MX (i.e. under conditions where single-nucleotide BER is blocked) the null cells are still considerably more sensitive than wild-type. The data are consistent with a role of beta-pol in long patch BER, which helps protect cells against methylation damage-induced cytotoxicity.

Alkylating Agents↗
PubMed2000

Molecular cloning and high-level expression of human polymerase beta cDNA and comparison of the purified recombinant human and rat enzymes.

The cDNA encoding the human polymerase beta from HeLa cells was PCR amplified and cloned, and its nucleotide sequence determined. The DNA sequence is identical to the polymerase beta cDNA sequence from Tera-2 cells. Three expression strategies were employed that were designed to maximize translation initiation of the polymerase beta mRNA in Escherichia coli and all yielded a high level of human polymerase beta. The recombinant protein was purified and its properties were compared with those of the recombinant rat enzyme. The domain structure and kinetic parameters (k(cat) and K(m)) were nearly identical. A mouse IgG monoclonal antibody to the rat enzyme (mAb-10S) was approximately 10-fold less reactive with the human enzyme than with the rat enzyme as determined by ELISA.

Amino Acid Sequence↗
PubMed2000

Activated nuclear factor-kappaB is present in the coronary vasculature in experimental hypercholesterolemia.

BACKGROUND: Experimental hypercholesterolemia (HC) is characterized by a decrease in nitric oxide (NO) bioavailability and cellular proliferation. Nuclear factor-kappaB (NF-kappaB) is a transcriptional factor which plays a coordinating role in inflammation and cellular proliferation and may be involved in early atherosclerosis. We examined whether activated NF-kappaB was present in experimental hypercholesterolemia in the coronary vasculature in association with a decrease in NO bioavailability. METHODS: A total of 14 juvenile domestic crossbred pigs were placed on a HC diet and six pigs on a normal diet for 10-12 weeks. A monoclonal antibody to the activated form of the p65 subunit of NF-kappaB was used to detect immunoreactivity in coronary artery sections. Coronary tissue homogenates were analyzed for activated NF-kappaB and endothelial nitric oxide synthase (eNOS) using Western blotting. In vitro coronary endothelium-dependent relaxation was performed in response to bradykinin, as a measure of NO bioavailability. RESULTS: Intimal staining for activated NF-kappaB was present in 12/14 HC pigs as compared with 0/6 controls (P<0.001). Confocal microscopy confirmed the presence of NF-kappaB in the nucleus of intimal cells although the majority of the staining was cytoplasmic. In the HC group, Western blotting revealed an increase in activated NF-kappaB in the vessel wall compared to the normal group, in association with a decrease in the presence of eNOS protein and an attenuated vasorelaxation response to bradykinin. CONCLUSION: This study suggests a potential role for activation of NF-kappaB, in association with a decrease in NO bioavailability, in the initial stages of atherosclerosis in the coronary vasculature.

Animals↗
PubMed2000

Modulation of base excision repair by low density lipoprotein, oxidized low density lipoprotein and antioxidants in mouse monocytes.

In the present study, we found that oxidized low density lipoprotein, but not low density lipoprotein, down-regulated base excision repair activity in extracts of mouse monocyte cell line PU5-1.8. An enzyme required in this pathway, DNA polymerase beta, was also down-regulated. In contrast, treatment of monocytes with a combination of ascorbate and alpha-tocopherol up-regulated base excision repair activity and expression of DNA polymerase beta. Co-treatment of monocytes with antioxidants plus oxidized low density lipoprotein prevented down-regulation by oxidized low density lipoprotein. Oxidative DNA damage, as measured by 8-hydroxyguanine accumulation in genomic DNA, was found in cells treated with oxidized low density lipoprotein; 8-hydroxyguanine was not found in the cells treated with low density lipoprotein, antioxidants or oxidized low density lipoprotein plus antioxidants. These results establish a linkage between the DNA base excision repair pathway, oxidative DNA damage and oxidized low density lipoprotein treatment in mouse monocytes. Since oxidized low density lipoprotein is implicated in chronic disease conditions such as atherogenesis, these findings facilitate understanding of genetic toxicology mechanisms related to human health and disease.

Animals↗
PubMed2000

Biomedical research leaders: report on needs, opportunities, difficulties, education and training, and evaluation.

The National Association of Physicians for the Environment (NAPE) has assumed a leadership role in protecting environmental health in recent years. The Committee of Biomedical Research Leaders was convened at the recent NAPE Leadership Conference: Biomedical Research and the Environment held on 1--2 November 1999, at the National Institutes of Health, Bethesda, Maryland. This report summarizes the discussion of the committee and its recommendations. The charge to the committee was to raise and address issues that will promote and sustain environmental health, safety, and energy efficiency within the biomedical community. Leaders from every important research sector (industry laboratories, academic health centers and institutes, hospitals and care facilities, Federal laboratories, and community-based research facilities) were gathered in this committee to discuss issues relevant to promoting environmental health. The conference and this report focus on the themes of environmental stewardship, sustainable development and "best greening practices." Environmental stewardship, an emerging theme within and outside the biomedical community, symbolizes the effort to provide an integrated, synthesized, and concerted effort to protect the health of the environment in both the present and the future. The primary goal established by the committee is to promote environmentally responsible leadership in the biomedical research community. Key outcomes of the committee's discussion and deliberation were a) the need for a central organization to evaluate, promote, and oversee efforts in environmental stewardship; and b) immediate need to facilitate efficient information transfer relevant to protecting the global environment through a database/clearinghouse. Means to fulfill these needs are discussed in this report.

Biomedical Technology↗
PubMed2000

Decreasing mortality with primary percutaneous coronary intervention in patients with acute myocardial infarction: the Mayo Clinic experience from 1991 through 1997.

OBJECTIVE: To characterize and determine the overall impact of changes in primary percutaneous coronary intervention (PCI) on the clinical outcome of patients presenting within 24 hours of acute myocardial infarction (AMI). PATIENTS AND METHODS: We retrospectively analyzed a prospective PCI registry for 1073 consecutive patients undergoing primary PCI for AMI at the Mayo Clinic in Rochester, Minn, from 1991 through 1997. The primary outcome measure was mortality from any cause within 30 days and 1 year. RESULTS: The number of patients treated for AMI by primary PCI per year increased from 119 in 1991 to 193 in 1997. Intracoronary stent use increased from 1.7% in 1991 to 64.8% in 1997 (P < .001). This coincided with an increase in ticlopidine use from 3.6% in 1994 to 62.1% in 1997 (P < .001) and in abciximab use from 2.7% in 1995 to 63.2% in 1997 (P < .001). An increase in beta-blocker (58.3% to 75.3%; P < .001), angiotensin-converting enzyme inhibitor (0.9% to 40.0%; P < .001), and 3-hydroxy-3-methylglutaryl coenzyme A reductase use (1.9% to 40.5%; P < .001) as well as a decrease in calcium channel antagonist (34.3% to 8.4%; P < .001) use occurred on discharge. From 1991 through 1997, there was a significant decrease in the 30-day mortality rate (10.1% to 5.2%; P = .05). The 1-year mortality rate also decreased (13.4% in 1991 to 10.4% in 1997) (P = .09). After adjustment for other confounding variables, treatment in more recent years was associated with a significant decrease in death at 30 days (odds ratio, 0.89; 95% confidence interval, 0.79-1.00; P = .05) and during long-term follow-up (odds ratio, 0.93; 95% confidence interval, 0.87-1.00; P = .04). CONCLUSIONS: Percutaneous coronary intervention methods of reperfusion for AMI, along with adjuvant pharmacotherapy, have changed over recent years and have been associated with improved short- and long-term survival.

Abciximab↗
PubMed1999

Uniquely altered DNA replication fidelity conferred by an amino acid change in the nucleotide binding pocket of human immunodeficiency virus type 1 reverse transcriptase.

Arginine 72 in human immunodeficiency virus type 1 reverse transcriptase (RT), a highly conserved residue among retroviral polymerases and telomerases, forms part of the binding pocket for the nascent base pair. We show here that replacement of Arg(72) by alanine strongly alters fidelity in a highly unusual manner. R72A reverse transcriptase is a frameshift and base substitution antimutator polymerase whose increased fidelity results both from increased nucleotide selectivity and from a decreased ability to extend mismatched primer termini. Thus, Arg(72)-substrate interactions in wild-type human immunodeficiency virus type 1 RT can stabilize incorrect nucleotides allowing misinsertion and promoting extension of mismatched and perhaps misaligned template-primers. In contrast to the higher fidelity at most sites, R72A RT is highly error-prone for misincorporations opposite template T in the sequence context: 5'-CTGG. Surprisingly, this results mostly from a 1200-fold increase in the apparent K(m) for correct dAMP incorporation. Thus, Arg(72) interactions with substrate are critical for the stability of the correct T.dAMP base pair when the 5'-CTGG sequence is present in the binding pocket for the nascent base pair. Collectively, the data show that a mutant polymerase may yield higher than normal average replication fidelity, yet paradoxically place specific sequences at very high risk of mutation.

Base Pairing↗
PubMed1999

Base substitution specificity of DNA polymerase beta depends on interactions in the DNA minor groove.

To examine the hypothesis that interactions between a DNA polymerase and the DNA minor groove are critical for accurate DNA synthesis, we studied the fidelity of DNA polymerase beta mutants at residue Arg(283), where arginine, which interacts with the minor groove at the active site, is replaced by alanine or lysine. Alanine substitution, removing minor groove interactions, strongly reduces polymerase selectivity for all single-base mispairs examined. In contrast, the lysine substitution, which retains significant interactions with the minor groove, has wild-type-like selectivity for T.dGMP and A.dGMP mispairs but reduced selectivity for T.dCMP and A.dCMP mispairs. Examination of DNA crystal structures of these four mispairs indicates that the two mispairs excluded by the lysine mutant have an atom (N2) in an unfavorable position in the minor groove, while the two mispairs permitted by the lysine mutant do not. These results suggest that unfavorable interactions between an active site amino acid side chain and mispair-specific atoms in the minor groove contribute to DNA polymerase specificity.

Alanine↗
PubMed1999

Residues in the alphaH and alphaI helices of the HIV-1 reverse transcriptase thumb subdomain required for the specificity of RNase H-catalyzed removal of the polypurine tract primer.

During retrovirus replication, reverse transcriptase (RT) must specifically interact with the polypurine tract (PPT) to generate and subsequently remove the RNA primer for plus-strand DNA synthesis. We have investigated the role that human immunodeficiency virus-1 RT residues in the alphaH and alphaI helices in the thumb subdomain play in specific RNase H cleavage at the 3'-end of the PPT; an in vitro assay modeling the primer removal step was used. Analysis of alanine-scanning mutants revealed that a subgroup exhibits an unusual phenotype in which the PPT is cleaved up to seven bases from its 3'-end. Further analysis of alphaH mutants (G262A, K263A, N265A, and W266A) with changes in residues in or near a structural motif known as the minor groove binding track showed that the RNase H activity of these mutants is more dramatically affected with PPT substrates than with non-PPT substrates. Vertical scan mutants at position 266 were all defective in specific RNase H cleavage, consistent with conservation of tryptophan at this position among lentiviral RTs. Our results indicate that residues in the thumb subdomain and the minor groove binding track in particular, are crucial for unique interactions between RT and the PPT required for correct positioning and precise RNase H cleavage.

Alanine↗
PubMed1999

"Action-at-a-distance" mutagenesis. 8-oxo-7, 8-dihydro-2'-deoxyguanosine causes base substitution errors at neighboring template sites when copied by DNA polymerase beta.

8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG), a common oxidative DNA lesion, favors a syn-conformation in DNA, enabling formation of stable 8-oxo-dG.A base mispairs resulting in G.C --> T.A transversion mutations. When human DNA polymerase (pol) beta was used to copy a short single-stranded gap containing a site-directed 8-oxo-dG lesion, incorporation of dAMP opposite 8-oxo-dG was slightly favored over dCMP depending on "downstream" sequence context. Unexpectedly, however, a significant increase in dCMP.A and dGMP.A mispairs was also observed at the "upstream" 3'-template site adjacent to the lesion. Errors at these undamaged template sites occurred in four sequence contexts with both gapped and primed single-stranded DNA templates, but not when pol alpha replaced pol beta. Error rates at sites adjacent to 8-oxo-dG were roughly 1% of the values opposite 8-oxo-dG, potentially generating tandem mutations during in vivo short-gap repair synthesis by pol beta. When 8-oxo-dG was replaced with 8-bromo-2'-deoxyguanosine, incorporation of dCMP was strongly favored by both enzymes, with no detectable misincorporation occurring at neighboring template sites.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1999

Role of DNA polymerase beta in the excision step of long patch mammalian base excision repair.

The two base excision repair (BER) subpathways in mammalian cells are characterized by the number of nucleotides synthesized into the excision patch. They are the "single-nucleotide" BER pathway and the "long patch" (several nucleotides incorporated) BER pathway. Both of these subpathways involve excision of a damaged base and/or nearby nucleotides and DNA synthesis to fill the excision gap. Whereas DNA polymerase beta (pol beta) is known to participate in the single-nucleotide BER pathway, the identity of polymerases involved in long patch BER has remained unclear. By analyzing products of long patch excision generated during BER of a uracil-containing DNA substrate in mammalian cell extracts we find that long patch excision depends on pol beta. We show that the excision of the characteristic 5'-deoxyribose phosphate containing oligonucleotide (dRP-oligo) is deficient in extracts from pol beta null cells and is rescued by addition of purified pol beta. Also, pol beta-neutralizing antibody inhibits release of the dRP-oligo in wild-type cell extracts, and the addition of pol beta after inhibition with antibody completely restores the excision reaction. The results indicate that pol beta plays an essential role in long patch BER by conducting strand displacement synthesis and controlling the size of the excised flap.

Animals↗
PubMed1999

Timing of coronary stent thrombosis in patients treated with ticlopidine and aspirin.

In patients receiving coronary stents treated with aspirin and coumadin, the peak incidence of stent thrombosis occurs on the fifth and sixth days following the implantation procedure. Little is known about the timing of stent thrombosis in patients treated with aspirin and ticlopidine. We compared the timing of coronary stent thrombosis in patients treated with ticlopidine and aspirin with the timing in those receiving coumadin and aspirin. A retrospective databank analysis was performed and 39 patients were identified who experienced stent thrombosis after successful coronary stent implantation. Of these, 21 had been treated with ticlopidine and aspirin and 18 with coumadin and aspirin therapy. The median time from stent implantation to stent thrombosis in the ticlopidine and aspirin group was 12 hours (interquartile range 6 to 72 hours) compared with 4 days in the coumadin and aspirin group (interquartile range 21 to 68 hours) (p <0.0001). There was no significant difference between the timing of stent thrombosis in patients treated with abciximab in addition to ticlopidine and aspirin (median 17 hours, interquartile range 6 to 29) versus ticlopidine and aspirin patients who did not receive abciximab (median 11 hours, interquartile range 9 to 12, p = 0.57). Thus, in patients who receive coronary stents, stent thrombosis occurs much earlier after the procedure in patients treated with ticlopidine and aspirin than in patients treated with anticoagulation therapy.

Angioplasty, Balloon, Coronary↗
PubMed1999

Cells deficient in DNA polymerase beta are hypersensitive to alkylating agent-induced apoptosis and chromosomal breakage.

DNA polymerase beta (beta-pol), which is involved in base excision repair, was investigated for its role in protection of cells against various genotoxic agents and cytostatic drugs using beta-pol knockout mouse fibroblasts. We show that cells lacking beta-pol are highly sensitive to induction of apoptosis and chromosomal breakage by methylating agents, such as N-methyl-N'-nitro-N-nitrosoguanidine and methyl methanesulfonate and the cross-linking antineoplastic drugs mitomycin C and mafosfamide. The cross-sensitivity between the agents observed suggests that beta-pol is involved in repair not only of DNA methylation lesions but also of other kinds of DNA damage induced by various cytostatic drugs. Cells deficient in beta-pol were not hypersensitive to cisplatin, melphalan, benzo(a)pyrene diol epoxide, chloroethylnitrosourea, or UV light. Because both established and primary beta-pol knockout fibroblasts displayed the hypersensitive phenotype, which, moreover, was complemented by transfection with a beta-pol expression vector, the alkylating agent hypersensitivity can clearly be attributed to the beta-pol deficiency. The results demonstrate that beta-pol-driven base excision repair is highly important for protection of cells against cell killing due to apoptosis and induced chromosomal breakage and suggest that incompletely repaired DNA damage causes chromosomal changes and may act as a trigger of DNA damage-induced apoptosis.

Alkylating Agents↗