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Biomedical subjects

S H Wilson

Publications and source records attributed to S H Wilson.

At least 91 records · Page 5Linked to original sources
PubMed1998

Neointimal response following rotational atherectomy compared to balloon angioplasty in a porcine model of coronary in-stent restenosis.

In-stent restenosis remains a clinical therapeutic challenge. Rotational atherectomy (RA) is an attractive treatment option as it may cause less vascular injury than balloon angioplasty (BA) and, therefore, limit further neointimal response. In an animal model of coronary in-stent restenosis, thermal injury and stenting created neointima (old NI). The treatment of in-stent restenosis with either BA (n = 9) or RA (n = 11) also generated neointima (new NI). The average areas (mm2) of old NI in the BA and RA groups were similar (3.77 +/- 0.40 vs. 3.67 +/- 0.53; P = 0.32). However, new NI formed after treatment of in-stent restenosis was significantly less in the RA as compared to the BA group (0.33 +/- 0.12 vs. 0.73 +/- 36, P < 0.01). In this porcine coronary artery model of in-stent restenosis, treatment with rotational atherectomy resulted in significantly less recurrent neointimal hyperplasia than balloon angioplasty. This animal study, thus, provides a rationale for the clinical use of rotablation in the treatment of in-stent restenosis.

Angioplasty, Balloon, Coronary↗
PubMed1998

The mental and physical health of miners following the 1992 national pit closure programme: a cross sectional survey using General Health Questionnaire GHQ-12 and Short Form SF-36.

BACKGROUND: In the twelve months following the announcement of the UK pit closure programme in October 1992, 22,500 miners were made redundant. In 1994 we undertook a cross-sectional survey to determine whether the mental and physical health of men who had been employed in the Nottinghamshire mining industry differed from that of the general population. METHODS: A postal questionnaire was designed incorporating the General Health Questionnaire (GHQ-12), and six domains from SF-36. Questionnaires were sent to 1064 miners and ex-miners and 2097 other men in Nottinghamshire. Non-responders were sent two reminders. RESULTS: The final response rate was 51%. The percentage of responders with GHQ-12 scores of three or more (suggesting psychological disorder) was 46% for those still employed in the mining industry, 52% for unemployed former miners and 22% for working non-miners (odds ratios: 3.0 [95% C.I. 2.2-4.1] for current miners and 3.9 [95% C.I. 2.6-5.7] for unemployed miners compared with working non-miners). The miners and ex-miners also had lower scores (suggesting greater morbidity) for each of the SF-36 domains tested. When stratifying for age in respondents of social classes IIIM-V the scores of current miners were significantly lower than those of working non-miners (P < 0.01). CONCLUSIONS: This study suggests that when surveyed in 1994, men who had been employed in three Nottinghamshire collieries in 1992 were psychologically and physically disadvantaged compared with working non-miners. Whether these findings are a result of pit closures is uncertain. However, significant potential health needs have been demonstrated.

Adolescent↗
PubMed1997

Crystal structures of human DNA polymerase beta complexed with gapped and nicked DNA: evidence for an induced fit mechanism.

DNA polymerase beta (pol beta) fills single nucleotide (nt) gaps in DNA produced by the base excision repair pathway of mammalian cells. Crystal structures have been determined representing intermediates in the 1 nt gap-filling reaction of pol beta: the binary complex with a gapped DNA substrate (2.4 A resolution), the ternary complex including ddCTP (2.2 A), and the binary product complex containing only nicked DNA (2.6 A). Upon binding ddCTP to the binary gap complex, the thumb subdomain rotates into the closed conformation to contact the otherwise solvent-exposed ddCTP-template base pair. Thumb movement triggers further conformational changes which poise catalytic residue Asp192, dNTP, and template for nucleotidyl transfer, effectively assembling the active site. In the product nicked DNA complex, the thumb returns to the open conformation as in the gapped binary DNA complex, facilitating dissociation of the product. These findings suggest that pol beta may enhance fidelity by an induced fit mechanism in which correct base pairing between template and incoming dNTP induces alignment of catalytic groups for catalysis (via thumb closure), but incorrect base pairing will not. The structures also reveal that pol beta binds both gapped and nicked DNA with a 90 degrees kink occurring precisely at the 5'-phosphodiester linkage of the templating residue. If the DNA were not kinked in this way, contact between the thumb and dNTP-template base pair, presumably important for the checking mechanism, would be impossible, especially when the gap is but a single nucleotide. Such a 90 degrees kink may be a mechanistic feature employed by any polymerase involved in filling gaps to completion.

Catalysis↗
PubMed1997

Identification of N(G)-methylarginine residues in human heterogeneous RNP protein A1: Phe/Gly-Gly-Gly-Arg-Gly-Gly-Gly/Phe is a preferred recognition motif.

Three sites of N(G),N(G)-arginine methylation have been located at residues 205, 217, and 224 in the glycine-rich, COOH-terminal one-third of the HeLa A1 heterogeneous ribonucleoprotein. Together with the previously determined dimethylated arginine at position 193 [Williams et al., (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 5666-5670], it is evident that all four sites fall within a span of sequence between residues 190 and 233 that contains multiple Arg-Gly-(Gly) sequences interspersed with phenylalanine residues. These RGG boxes have been postulated to represent an RNA binding motif [Kiledjian and Dreyfuss (1992) EMBO J. 11, 2655-2664]. Dimethylation of HeLa A1 appears to be quantitative at each of the four positions. Arginines 205 and 224 have been methylated in vitro by a nuclear protein arginine methyltransferase using recombinant (unmethylated) A1 as substrate. This suggests A1 may be an in vivo substrate for this enzyme. Examination of sequences surrounding the sites of methylation in A1 along with a compilation from the literature of sites that have been identified in other nuclear RNA binding proteins suggests a methylase-preferred recognition sequence of Phe/Gly-Gly-Gly-Arg-Gly-Gly-Gly/Phe, with the COOH-terminal flanking glycine being obligatory. Taken together with data in the literature, identification of the sites of A1 arginine methylation strongly suggests a role for this modification in modulating the interaction of A1 with nucleic acids.

Amino Acid Sequence↗
PubMed1997

Probing structure/function relationships of HIV-1 reverse transcriptase with styrene oxide N2-guanine adducts.

Details of the interactions between the human immunodeficiency virus (HIV-1) reverse transcriptase and substrate DNA were probed both by introducing site-specific and stereospecific modifications into DNA and by altering the structure of potential critical residues in the polymerase. Unadducted 11-mer DNAs and 11-mer DNAs containing R and S enantiomers of styrene oxide at N2-guanine were ligated with two additional oligonucleotides to create 63-mers that served as templates for HIV-1 reverse transcriptase replication. Oligonucleotides that primed synthesis 5 bases 3' to the adducts could be extended up to 1 base 3' and opposite the lesion. However, when the positions of the 3'-OH of the priming oligonucleotides were placed 1, 2, 3, 4, 5, and 6 bases downstream of the styrene oxide guanine adducts, replication was initiated, only to be blocked after incorporating 4, 5, 6, and 7 bases beyond the lesion. The sites of this adduct-induced termination corresponded to the position of the DNA where alpha-helix H makes contact with the DNA minor groove, 3-5 bases upstream of the growing 3' end. In addition, mutants of the polymerase in alpha-helix H (W266A and G262A) alter the termination probabilities caused by these DNA adducts, suggesting that alpha-helix H is a sensitive monitor of modifications in the minor groove of newly synthesized template-primer DNA several bases distal to the 3'-OH.

Base Sequence↗
PubMed1997

Abasic translesion synthesis by DNA polymerase beta violates the "A-rule". Novel types of nucleotide incorporation by human DNA polymerase beta at an abasic lesion in different sequence contexts.

The "A-rule" reflects the preferred incorporation of dAMP opposite abasic lesions in Escherichia coli in vivo. DNA polymerases (pol) from procaryotic and eucaryotic organisms incorporate nucleotides opposite abasic lesions in accordance with the A-rule. However, recent in vivo data demonstrate that A is not preferentially incorporated opposite abasic lesions in eucaryotes. Purified human DNA polymerases beta and alpha are used to measure the specificity of nucleotide incorporation at a site-directed tetrahydrofuran abasic lesion, in 8-sequence contexts, varying upstream and downstream bases adjacent to the lesion. Extension past the lesion is measured in 4 sequence contexts, varying the downstream template base. Pol alpha strongly favors incorporation of dAMP directly opposite the lesion. In marked contrast, pol beta violates the A-rule for incorporation directly opposite the lesion. In addition to incorporation taking place directly opposite the lesion, we also analyze misalignment incorporation directed by a template base downstream from the lesion. Lesion bypass by pol beta occurs predominantly by "skipping over" the lesion, by insertion of a nucleotide complementary to an adjacent downstream template site. Misalignment incorporation for pol beta occurs by a novel "dNTP-stabilized" mechanism resulting in both deletion and base substitution errors. In contrast, pol alpha shows no propensity for this type of synthesis. The misaligned DNA structures generated during dNTP-stabilized lesion bypass do not conform to misaligned structures reported previously.

Base Sequence↗
PubMed1997

Substrate binding by human apurinic/apyrimidinic endonuclease indicates a Briggs-Haldane mechanism.

Apurinic/apyrimidinic endonuclease (AP endo) makes a single nick 5' to a DNA abasic site. We have characterized this reaction by steady-state and transient-state kinetics with purified human AP endo, which had been expressed in Escherichia coli. The substrate was a 49-base pair oligonucleotide with an abasic site at position 21. This substrate was generated by treating a 49-mer duplex oligonucleotide with a single G/U located at position 21 with uracil-DNA glycosylase. The enzymatic products of the AP endo nicking reaction were a 20-mer with a hydroxyl group at the 3'-terminus and a 28-mer with a phosphodeoxyribose at the 5'-terminus. To obtain maximal enzymatic activity, it was necessary to stabilize the abasic site during treatment with uracil-DNA glycosylase with a reducing agent. Otherwise, a 20-mer with phosphoribose at the 3'-terminus resulted from beta-elimination. In agreement with others, Km and kcat were 100 nM and 10 s(-1), respectively. Heat treatment of the abasic site-containing 49-mer without enzyme also resulted in conversion to the beta-elimination product. The resultant heat degradation product was an efficient inhibitor of AP endo with a Ki of 30 nM. The enzyme required divalent cation (Mg2+) for activity, but bound substrate DNA in the absence of Mg2+. Electrophoretic mobility shift assays indicated that AP endo bound tightly to DNA containing an abasic site and formed a 1:1 complex at low enzyme concentrations. The association and dissociation rate constants for substrate binding to AP endo were determined by using a challenge assay to follow AP endo-substrate complex formation. Heat degradation product together with heparin served as an effective trap for free enzyme. The results are consistent with a Briggs-Haldane mechanism where k(on) and k(off) are 5 x 10(7) M(-1) s(-1) and 0.04 s(-1), respectively (Kd = 0.8 nM), kcat is 10 s(-1), and product release is very rapid (i.e. k(off,product) >> 10 s(-1)). This scheme is in excellent agreement with the measured steady-state kinetic parameters.

DNA-(Apurinic or Apyrimidinic Site) Lyase↗
PubMed1997

Disruption of transcription in vitro and gene expression in vivo by DNA adducts derived from a benzo[a]pyrene diol epoxide located in heterologous sequences.

Previous studies indicated a high affinity of the transcription factor Sp1 for DNA adducts derived from benzo[a]pyrene diol epoxide (BPDE) in sequences that are not normal binding sites for Sp1. We tested for functional effects of this phenomenon in three systems in which transcription is Sp1-dependent. In an in vitro, Sp1-dependent transcription system addition of heterologous plasmid DNA containing BPDE adducts abolished production of a specific run-off transcript. This inhibition was not seen with unmodified plasmid DNA, and could be overcome by addition of purified Sp1 protein. In SL2 insect cells, high-level expression of an Sp1-dependent reporter gene, which was dependent on co-transfection of an Sp1 expression vector, was inhibited >95% by co-transfection of heterologous DNA containing BPDE adducts. This inhibition could be partially overcome by increasing the amount of the Sp1 expression vector in the transfections. In human C33A cells, expression of a transfected reporter gene driven by a GC box containing fragment of the human E2F1 promoter was enhanced by co-transfection of an Sp1 expression plasmid. Expression was inhibited 3-6-fold by co-transfection of heterologous DNA containing BPDE-DNA adducts. A similar inhibition was seen in human SAOS-2 cells, which lack functional p53 protein. These data are consistent with functionally significant sequestration of the Sp1 transcription factor by BPDE-DNA adducts in all three systems. Altered availability of transcription factors such as Sp1 in carcinogen-treated cells may disrupt patterns of gene expression.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1996

Crystal structures of human DNA polymerase beta complexed with DNA: implications for catalytic mechanism, processivity, and fidelity.

Mammalian DNA polymerase beta (pol beta) is a small (39 kDa) DNA gap-filling enzyme that comprises an amino-terminal 8-kDa domain and a carboxy-terminal 31-kDa domain. In the work reported here, crystal structures of human pol beta complexed with blunt-ended segments of DNA show that, although the crystals belong to a different space group, the DNA is nevertheless bound in the pol beta binding channel in the same way as the DNA in previously reported structures of rat pol beta complexed with a template-primer and ddCTP [Pelletier, H., Sawaya, M. R., Kumar, A., Wilson, S. H., & Kraut, J. (1994) Science 264, 1891-1903]. The 8-kDa domain is in one of three previously observed positions relative to the 31-kDa domain, suggesting that the 8-kDa domain may assume only a small number of stable conformations. The thumb subdomain is in a more open position in the human pol beta-DNA binary complex than it is in the rat pol beta-DNA-ddCTP ternary complex, and a closing thumb upon nucleotide binding could represent the rate-limiting conformational change that has been observed in pre-steady-state kinetic studies. Intermolecular contacts between the DNA and the 8-kDa domain of a symmetry-related pol beta molecule reveal a plausible binding site on the 8-kDa domain for the downstream oligonucleotide of a gapped-DNA substrate; in addition to a lysine-rich binding pocket that accommodates a 5'-PO4 end group, the 8-kDa domain also contains a newly discovered helix-hairpin-helix (HhH) motif that binds to DNA in the same way as does a structurally and sequentially homologous HhH motif in the 31-kDa domain. DNA binding by both HhH motifs is facilitated by a metal ion. In that HhH motifs have been identified in other DNA repair enzymes and DNA polymerases, the HhH-DNA interactions observed in pol beta may be applicable to a broad range of DNA binding proteins. The sequence similarity between the HhH motif of endonuclease III from Escherichia coli and the HhH motif of the 8-kDa domain of pol beta is particularly striking in that all of the conserved residues are clustered in one short sequence segment, LPGVGXK, where LPGV corresponds to a type II beta-turn (the hairpin turn), and GXK corresponds to a part of the HhH motif that is proposed to be critical for DNA binding and catalysis for both enzymes. These results suggest that endonuclease III and the 8-kDa domain of pol beta may employ a similar mode of DNA binding and may have similar catalytic mechanisms for their respective DNA lyase activities. A model for productive binding of pol beta to a gapped-DNA substrate requires a 90 degrees bend in the single-stranded template, which could enhance nucleotide selectivity during DNA repair or replication.

Amino Acid Sequence↗
PubMed1996

A structural basis for metal ion mutagenicity and nucleotide selectivity in human DNA polymerase beta.

When crystals of human DNA polymerase beta (pol beta) complexed with DNA [Pelletier, H., Sawaya, M. R., Wolfle, W., Wilson, S. H., & Kraut, J. (1996) Biochemistry 35, 12742-12761] are soaked in the presence of dATP and Mn2+, X-ray structural analysis shows that nucleotidyl transfer to the primer 3'-OH takes place directly in the crystals, even though the DNA is blunt-ended at the active site. Under similar crystal-soaking conditions, there is no evidence for a reaction when Mn2+ is replaced by Mg2+, which is thought to be the divalent metal ion utilized by most polymerases in vivo. These results suggest that one way Mn2+ may manifest its mutagenic effect on polymerases is by promoting greater reactivity than Mg2+ at the catalytic site, thereby allowing the nucleotidyl transfer reaction to take place with little or no regard to instructions from a template. Non-template-directed nucleotidyl transfer is also observed when pol beta-DNA cocrystals are soaked in the presence of dATP and Zn2+, but the reaction products differ in that the sugar moiety of the incorporated nucleotide appears distorted or otherwise cleaved, in agreement with reports that Zn2+ may act as a polymerase inhibitor rather than as a mutagen [Sirover, M. A., & Loeb, L. A. (1976) Science 194, 1434-1436]. Although no reaction is observed when crystals are soaked in the presence of dATP and other metal ions such as Ca2+, Co2+, Cr3+, or Ni2+, X-ray structural analyses show that these metal ions coordinate the triphosphate moiety of the nucleotide in a manner that differs from that observed with Mg2+. In addition, all metal ions tested, with the exception of Mg2+, promote a change in the side-chain position of aspartic acid 192, which is one of three highly conserved active-site carboxylate residues. Soaking experiments with nucleotides other than dATP (namely, dCTP, dGTP, dTTP, ATP, ddATP, ddCTP, AZT-TP, and dATP alpha S) reveal a non-base-specific binding site on pol beta for the triphosphate and sugar moieties of a nucleotide, suggesting a possible mechanism for nucleotide selectivity whereby triphosphate-sugar binding precedes a check for correct base pairing with the template.

Carcinogens↗
PubMed1996

dNTP binding to HIV-1 reverse transcriptase and mammalian DNA polymerase beta as revealed by affinity labeling with a photoreactive dNTP analog.

The dNTP binding pocket of human immunodeficiency virus type 1 reverse transcriptase (RT) and DNA polymerase beta (beta-pol) were labeled using a photoreactive analog of dCTP, exo-N-[beta-(p-azidotetrafluorobenzamido)-ethyl]-deoxycytidine-5'- triphosphate (FABdCTP). Two approaches of photolabeling were utilized. In one approach, photoreactive FABdCTP and radiolabeled primer-template were UV-irradiated in the presence of each enzyme and resulted in polymerase radiolabeling. In an alternate approach, FABdCTP was first UV-cross-linked to enzyme; subsequently, radiolabeled primer-template was added, and the enzyme-linked dCTP analog was incorporated onto the 3'-end of the radiolabeled primer. The results showed strong labeling of the p66 subunit of RT, with only minor labeling of p51. No difference in the intensity of cross-linking was observed with either approach. FABdCTP cross-linking was increased in the presence of a dideoxyterminated primer-template with RT, but not with beta-pol, suggesting a significant influence of prior primer-template binding on dNTP binding for RT. Mutagenesis of beta-pol residues observed to interact with the incoming dNTP in the crystal structure of the ternary complex resulted in labeling consistent with kinetic characterization of these mutants and indicated specific labeling of the dNTP binding pocket.

Affinity Labels↗
PubMed1996

Replication of O6-methylguanine-containing DNA by repair and replicative DNA polymerases.

The biological consequences of O6-methylguanine (m6G) in DNA are well recognized. When template m6G is encountered by DNA polymerases, replication is hindered and trans-lesion replication results in the preferential incorporation of dTMP opposite template m6G. Thus, unrepaired m6G in DNA is both cytotoxic and mutagenic. Yet, cell lines tolerant to m6G in DNA have been isolated, which indicates that some cellular DNA polymerases may replicate m6G-containing DNA with reasonable efficiency. Previous reports suggested that mammalian pol beta could not replicate m6G-containing DNA, but we find that pol beta can catalyze trans-lesion replication; however, the lesion must reside in the optimal context for pol beta activity, single- or short nucleotide gapped substrates. Primed single-stranded DNA templates, with or without template m6G, were poor substrates for pol beta as reported in earlier studies. In contrast, trans-lesion replication by bacteriophage T4 DNA polymerase was observed for primed single-stranded DNA templates. Replication of m6G-containing DNA by T4 DNA polymerase required the gp45 accessory protein that clamps the polymerase to the DNA template. The rate-limiting step in replicating m6G-containing DNAs by both DNA polymerases tested was incorporation of dTMP across from the lesion.

Base Sequence↗