Biomedical subjects

S H Wilson

Publications and source records attributed to S H Wilson.

At least 181 records · Page 10Linked to original sources

PubMed1990

Studies of the domain structure of mammalian DNA polymerase beta. Identification of a discrete template binding domain.

Characterization of the domain structure of DNA polymerase beta is reported. Large scale overproduction of the rat protein in Escherichia coli was achieved, and the purified recombinant protein was verified by sequencing tryptic peptides. This protein is both a single-stranded DNA binding protein and a DNA polymerase consisting of one polypeptide chain of 334 amino acids. As revealed by controlled proteolysis experiments, the protein is organized in two relatively protease-resistant segments linked by a short protease-sensitive region. One of these protease-resistant segments represents the NH2-terminal 20% of the protein. This NH2-terminal domain (of about 75 residues) has strong affinity for single-stranded nucleic acids. The other protease-resistant segment, representing the COOH-terminal domain of approximately 250 residues, does not bind to nucleic acids. Neither domain, tested as purified proteins, has substantial DNA polymerase activity. The results suggest that the NH2-terminal domain is principally responsible for the template binding activity of the intact protein.

Amino Acid Sequence↗

PubMed1990

Deregulation of DNA polymerase beta by sense and antisense RNA expression in mouse 3T3 cells alters cell growth.

DNA polymerase beta (beta-pol) and its mRNA are maintained at constitutive levels during the cell cycle and during stages of cell growth in culture. To study biological consequences of variations in the level of this DNA repair enzyme and/or its mRNA, we prepared expression vectors in which cDNA for human beta-pol is inserted under the control of a metallothionein promoter (pMT) in the sense and antisense orientation, respectively, and these vectors then were used for stable transformation of mouse 3T3 cells. Vectors also contained the mouse DHFR gene, such that culture of transformants in medium with increasing concentrations of methotrexate resulted in amplification of inserted DNA. The levels of sense and antisense transcripts are strongly increased by culture of transformants in medium with 65 microM Zn2+, although some expression is detected even without Zn2+ induction. After five days of induction, the beta-pol level was about threefold higher in sense cells and about 10-fold lower in antisense cells than in parallel cultures without induction. The antisense line has a threefold increased cell doubling time in the presence of 65 microM Zn2+ compared with the absence of Zn2+. Zn2+ (65 microM) induction for the sense line results in normal growth for the first three days and, thereafter, a complete cessation of growth. Yet, these blocked cells remain fully viable. The results indicate that sudden deregulation of beta-pol expression alters cell growth in mouse 3T3 cells.

Animals↗

PubMed1990

The social and demographic characteristics of women dying from cervical cancer in Nottingham.

Cervical cancer accounted for 57 deaths to women in Nottingham District in 1986 and 1987. 28% were women aged under 50 years of age. Using age specific death rates, the greatest risk was among women aged 60-69 years. However, when expressed as a proportion of all deaths, cervical cancer represents 8.5% of deaths in women aged 25-45, but only 0.6% of deaths to women over 46. There was no significant difference in relative risk between the five social classes, even when social class V was compared to aggregated data for social classes I-IV. This supports other evidence that the social class gradient in cervical cancer mortality is lessening. There was no excess risk shown to women resident in the inner city or in areas of social deprivation. This finding is in contrast to the work of others who have shown an increased incidence in areas of social deprivation. The evidence from this study would support those who suggested over 20 years ago that it is not social deprivation or occupation that constitute an increased risk to women, but sexual behaviour and other habits such as smoking.

Adult↗

PubMed1990

Potential for two isoforms of the A1 ribonucleoprotein in Xenopus laevis.

We have identified the Xenopus cognates for the RNA-binding protein A1. This protein has previously been shown to be one of the components of the heterogeneous nuclear ribonucleoprotein (hnRNP) complex in rat and human cells. We have isolated several Xenopus clones from oocyte, tailbud embryo, and leg muscle cDNA libraries and determined their nucleotide sequences. Potentially, two different A1 isoforms are expressed in Xenopus; they have been termed XA1a and XA1b. Besides insertions and deletions, the XA1a protein sequence is 92% identical to the rat protein and suggests very similar secondary structures. There are two segments in the COOH-terminal domain where deletions or insertions are apparent: the rat protein does not have a 48-residue sequence that is present in the frog protein, and the frog protein does not have a 12-residue sequence that is present in the rat protein. We have confirmed that the XA1a protein is larger than rat A1 by in vitro transcription, translation, and gel electrophoresis. The second isoform, XA1b, is very similar to the XA1a isoform, except it has a different COOH terminus due to the absence of a 73-nucleotide region from its cDNA clones. Transcripts representing both isoforms have been detected in various Xenopus RNA preparations by polymerase chain reaction experiments with A1-specific oligonucleotides. Our findings suggest that the isoforms are encoded by one or two genes and are the result of alternative splicing. We discuss the biological implications of having two forms of the A1 component of hnRNP particles.

Amino Acid Sequence↗

PubMed1990

Transfected human beta-polymerase promoter contains a ras-responsive element.

beta-Polymerase is a vertebrate cellular DNA polymerase involved in gap-filling synthesis during some types of genomic DNA repair. We report that a cloned human beta-polymerase promoter in a transient expression assay is activated by p21v-rasH expression in NIH 3T3 cells. A decanucleotide palindromic element, GTGACGTCAC, at positions -49 to -40 in the promoter is required for this ras-mediated stimulation.

Animals↗

PubMed1990

Mapping of the gene for DNA polymerase beta to mouse chromosome 8.

We conducted Southern blot analysis of DNAs from mouse x hamster somatic cell hybrids and progeny of an interspecies backcross to localize the mouse gene for DNA polymerase beta (Polb) to the centromeric end of Chromosome 8. These results provide additional support for the regional localization of polymerase beta on the short arm of human chromosome 8 or the very proximal end of the long arm.

Animals↗

PubMed1989

Specificity and mechanism of error-prone replication by human immunodeficiency virus-1 reverse transcriptase.

DNA-dependent DNA synthesis in vitro by human immunodeficiency virus-1 reverse transcriptase is relatively error-prone. The enzyme, whether recombinant or from virus particles, produces errors while replicating M13mp2 DNA at a rate that, if operative in vivo, would produce about five mutations per genome per round of replication. Sequence analysis of mutants resulting from in vitro synthesis demonstrates that errors are nonrandomly distributed. Base substitution and one-base frameshift mutational hot spots are observed. The specificity and position of errors suggest that most of the frameshifts and many of the base substitutions are initiated by template-primer slippage. Processivity analysis for the enzyme on the M13mp2 DNA template reveals strong termination at specific sites. Termination sites within homopolymer sequences correlate with frameshift mutational hot spots. The results suggest that the formation and/or utilization of misaligned template-primers is increased during the dissociation-reinitiation phase of the reaction.

Base Sequence↗

PubMed1989

Active-site modification of mammalian DNA polymerase beta with pyridoxal 5'-phosphate: mechanism of inhibition and identification of lysine 71 in the deoxynucleoside triphosphate binding pocket.

Pyridoxal 5'-phosphate is a potent inhibitor of the DNA polymerase activity of recombinant rat DNA polymerase beta. Kinetic studies indicate that the mechanism of PLP inhibition is complex. In a lower range of PLP concentration, inhibition is competitive with respect to substrate dNTP, whereas at higher levels of PLP several forms of enzyme combine with PLP and are involved in the overall inhibition, and a possible model for these interactions during the catalytic process is suggested. Reduction of the PLP-treated enzyme with sodium [3H]borohydride results in covalent incorporation of about 4 mol of PLP/mol of enzyme, and the modified enzyme is not capable of DNA polymerase activity. The presence of dNTP during the modification reaction blocks incorporation of 1 mol of PLP/mol of enzyme, and the enzyme so modified is almost fully active. This protective effect is not observed in the absence of template-primer. Tryptic peptide mapping of the PLP-modified enzyme reveals four major sites of modification. Of these four sites, only one is protected by dNTP from pyridoxylation. Sequence analysis of the tryptic peptide corresponding to the protected site reveals that it spans residues 68-80 in the amino acid sequence of the enzyme, with Lys 71 as the site of pyridoxylation. These results indicate that Lys 71 is at or near the binding pocket for the dNTP substrate.

Amino Acid Sequence↗

PubMed1989

Levels and size complexity of DNA polymerase beta mRNA in rat regenerating liver and other organs.

A cDNA probe encoding DNA polymerase beta (beta-pol) was used to study the level and size complexity of beta-pol mRNA in regenerating rat liver and other rat tissues. An almost 2-fold increase in beta-pol mRNA was observed 18-24 h after partial hepatectomy. In most adult rat tissues (liver, heart, kidney, stomach, spleen, thymus, lung and brain) the abundance of beta-pol mRNA was low. In contrast, young brain and testes exhibited beta-pol mRNA levels 5- and 15-times higher, respectively. The observed changes in the level of beta-pol mRNA in regenerating rat liver and in developing brain are correlated with reported changes in DNA polymerase beta activity. Four different (4.0, 2.5, 2.2, 1.4 kb) transcripts hybridizing to beta-pol probe were found in all tissues examined. The 4.0 kb transcript was dominant for young and adult brain, whereas the 1.4 kb transcript was dominant for testes. The significance of these transcripts is discussed.

Animals↗

PubMed1989

Stepwise mechanism of HIV reverse transcriptase: primer function of phosphorothioate oligodeoxynucleotide.

Primer recognition by purified HIV reverse transcriptase has been investigated. Earlier we found that the reaction pathway for DNA synthesis is ordered, with template-primer and free enzyme combining to form the first complex in the reaction sequence (Majumdar et al., 1988). We now find that d(C)28 is a linear competitive inhibitor of DNA synthesis against poly[r(A)].oligo[d(T)] as template.primer, indicating that d(C)28 and the template.primer combine with the same form of the enzyme in the reaction scheme, i.e., the free enzyme. The phosphorothioate oligodeoxynucleotide Sd(C)28 also is a linear competitive inhibitor against template.primer. However, the Ki for inhibition (approximately 2.8 nM) is approximately 200-fold lower than the Ki for inhibition by d(C)28. Since the inhibition is linear competitive, the dissociation constant is equal to the Ki for inhibition. Filter binding assays confirmed high-affinity binding between Sd(C)28 and the enzyme and yielded a KD similar to the Ki for inhibition. Substrate kinetic studies of DNA synthesis using Sd(C)28 as primer, and poly[r(I)] as template, revealed that the Km for Sd(C)28 is 24 nM. The Km for this primer is, therefore, 8-fold higher than the KD for enzyme-primer binding (2.8 nM). These results enable calculation of real time rate values for the enzyme-primer association (kon = 5.7 x 10(8) M-1 s-1) and dissociation (koff = 1.6 s-1).

Binding, Competitive↗

PubMed1989

Induction of beta-polymerase mRNA by DNA-damaging agents in Chinese hamster ovary cells.

Only a few of the genes involved in DNA repair in mammalian cells have been isolated, and induction of a DNA repair gene in response to DNA damage has not yet been established. DNA polymerase beta (beta-polymerase) appears to have a synthetic role in DNA repair after certain types of DNA damage. Here we show that the level of beta-polymerase mRNA is increased in CHO cells after treatment with several DNA-damaging agents.

Animals↗

PubMed1988

Human beta-polymerase gene. Structure of the 5'-flanking region and active promoter.

DNA polymerase beta (beta-pol) is a housekeeping enzyme considered to be involved in DNA repair in vertebrate cells. We cloned a fragment of genomic DNA spanning the first two exons of the human beta-pol gene and approximately 11 kilobases of the flanking region. The segment just 5' of the transcription start site can direct expression of the bacterial chloramphenicol acetyltransferase (CAT) gene in HeLa cells. A sequence containing only 113 base pairs of flanking DNA has promoter activity, and various constructs containing up to 4.8 kilobases of flanking sequence are expressed at a similar level, indicating that with this assay the important regulatory elements are located within or proximal to the approximately 100-bp core promoter. S1 nuclease mapping was used to show that transcription of the transfected genes is initiated at the same position as the endogenous beta-pol gene. The region upstream of the transcription start site is G + C rich and contains neither CAAT nor TATA boxes, but does have three decanucleotide elements matching high affinity binding sites for the RNA polymerase II transcription factor Sp1. Extending 5' from position -39 and surrounded by Sp1 consensus binding elements, there is a 10-nucleotide sequence with perfect dyad symmetry, GTGACGTCAC. Similar sequences are found in a number of cellular and viral promoters, including several adenovirus promoters. Experiments to test whether the core beta-pol promoter is activated by the adenovirus early region products showed that cotransfection with an adenovirus expression plasmid strongly activates expression of the beta-pol promoter.

Base Sequence↗

PubMed1988

Characterization of DNA polymerase beta mRNA: cell-cycle and growth response in cultured human cells.

DNA polymerase beta (beta-polymerase) is a housekeeping enzyme involved in DNA repair in vertebrate cells. We used a cDNA probe to study abundance of beta-polymerase mRNA in cultured human cells. The mRNA level in synchronized HeLa cells, representing different stages of the cell-cycle, varied only slightly. Contact inhibited fibroblasts AG-1522 contained the same level of mRNA as growing cells. The steady-state level of mRNA in fibroblasts is equivalent to 6 molecules per cell. The results indicate that the beta-polymerase transcript is "low abundance" and is neither cell-cycle nor growth phase responsive.

Cell Cycle↗

PubMed1988

Studies on the mechanism of human immunodeficiency virus reverse transcriptase. Steady-state kinetics, processivity, and polynucleotide inhibition.

A study of steady-state kinetics of polymerization by purified human immunodeficiency virus DNA polymerase (reverse transcriptase) has been conducted. DNA synthesis was examined using a system of poly(rA) as template, oligo(dT) as primer, and dTTP as nucleotide substrate. The substrate initial velocity patterns point to an ordered mechanism with template-primer adding first. Product inhibition kinetics with either pyrophosphate or phosphonoformate are consistent with this mechanism. The human immunodeficiency virus reverse transcriptase acts processively in this replication system, but exhibits some probability of terminating after each dTMP addition to the nascent chain. The probability of terminating was approximately 20-fold higher after the first dTMP addition than after subsequent additions. With this information on the mode of polymerization, appropriate kinetic models and steady-state rate equations are discussed. In further studies, we found that a heterologous polynucleotide, poly(rC), is a potent inhibitor of the enzyme. The pattern of this inhibition is uncompetitive against template-primer, suggesting that interaction with free enzyme is not the mechanism of the inhibition.

Algorithms↗

PubMed1988

Studies on the mechanism of Escherichia coli DNA polymerase I large fragment. Effect of template sequence and substrate variation on termination of synthesis.

Termination of Escherichia coli DNA polymerase I large fragment after processive synthesis on natural and other well-defined template.primer systems has been examined. We found that after any given deoxynucleoside monophosphate incorporation termination occurs in a nonrandom manner with phi X174 DNA as template: Termination is much more likely at some nucleotide residues along the template than at others. Analysis of these stronger termination sites indicates that the template base:incoming nucleotide combination influences termination. Introduction of a double-stranded region along the phi X174 template induces termination, and reducing dNTP concentrations or substituting 2'-deoxynucleoside 5'-O-(1-thio)triphosphate substrates also increases termination. Observations with the phi X174 DNA template system were extended with a defined template containing 1 inosine residue in an otherwise d(T)n homopolymer. Termination at the I residue is modulated by dCTP and decreases as dCTP concentration increases. A similar relationship is seen with the dCTP (1-thio) derivative, but termination is higher at given concentrations of this derivative than with dCTP. Pyrophosphate decreases general processivity in this system, but does not counteract the effect of increasing dCTP. Hill plot analysis of the dCTP effect in the inosine-containing template system gave a linear plot with Hill coefficient of 0.34, suggesting that dCTP influences termination at several steps in the polymerase reaction scheme. Substituting a methylated template base for I also increased termination, producing very strong blocks to processive synthesis. The results are consistent with a model in which termination occurs with several enzyme forms that are in equilibrium in an ordered catalytic mechanism.

Bacteriophage phi X 174↗

PubMed1988

Phenylalanines that are conserved among several RNA-binding proteins form part of a nucleic acid-binding pocket in the A1 heterogeneous nuclear ribonucleoprotein.

We have studied the domain structure of the A1 heterogeneous nuclear ribonucleoprotein using both partial proteolysis and photochemical cross-linking to oligodeoxynucleotides. Both the intact A1 protein and its proteolytic fragment, the UP1 protein, can be cleaved by Staphylococcus aureus V-8 protease to produce two polypeptides of 92 amino acids. These two polypeptides correspond to the internal repeat sequence previously noted by us to occur in UP1. The two polypeptides can be purified via single-stranded DNA cellulose chromatography and independently cross-linked to [32P]p(dT)8, indicating that each domain can bind to single-stranded nucleic acids. Purification and sequencing of A1 tryptic peptides that had been cross-linked to oligothymidylic acid revealed that 4 phenylalanine residues, phenylalanines 16, 58, 107, and 149 are the sites of covalent adduct formation, with phenylalanine 16 being the major site of cross-linking. These phenylalanine residues are internally homologous when the repeat sequences in A1 are aligned, that is, phenylalanines 16 and 107 occupy analogous positions in the 91-residue repeat, as do phenylalanines 58 and 149. An examination of the primary structures of a variety of eucaryotic RNA-binding proteins with sequence homology to A1 reveals that the cross-linked phenylalanines in A1 are highly conserved among all of these proteins. Our results provide the first experimental evidence that conserved residues in the 90-amino acid repeating domains shared by A1 and other single-stranded nucleic acid binding-proteins form part of an RNA-binding pocket.

Amino Acid Sequence↗

PubMed1988

Expression of human DNA polymerase beta in Escherichia coli and characterization of the recombinant enzyme.

The coding region of a human beta-polymerase cDNA, predicting a 335 amino acid protein, was subcloned in the Escherichia coli expression plasmid pRC23. After induction of transformed cells, the crude soluble extract was found to contain a new protein immunoreactive with beta-polymerase antibody and corresponding in size to the protein deduced from the cDNA. This protein was purified in a yield of 1-2 mg/50 g of cells. The recombinant protein had about the same DNA polymerase specific activity as beta-polymerase purified from mammalian tissues, and template-primer specificity and immunological properties of the recombinant polymerase were similar to those of natural beta-polymerases. The purified enzyme was free of nuclease activity. We studied detailed catalytic properties of the recombinant beta-polymerase using defined template-primer systems. The results indicate that this beta-polymerase is essentially identical with natural beta-polymerases. The recombinant enzyme is distributive in mode of synthesis and is capable of detecting changes in the integrity of the single-stranded template, such as methylated bases and double-stranded region. The enzyme recognizes a template region four to seven bases downstream of the primer 3' end and utilizes alternative primers if this downstream template region is double stranded. The enzyme is unable to synthesize past methylated bases N3-methyl-dT or O6-methyl-dG.

Amino Acid Sequence↗

PubMed1988

Mammalian heterogeneous nuclear ribonucleoprotein complex protein A1. Large-scale overproduction in Escherichia coli and cooperative binding to single-stranded nucleic acids.

Characterization of mammalian heterogeneous nuclear ribonucleoprotein complex protein A1 is reported after large-scale overproduction of the protein in Escherichia coli and purification to homogeneity. A1 is a single-stranded nucleic acid binding protein of 320 amino acids and 34,214 Da. The protein has two domains. The NH2-terminal domain is globular, whereas the COOH-terminal domain of about 120 amino acids has low probability of alpha-helix structure and is glycinerich. Nucleic acid binding properties of recombinant A1 were compared with those of recombinant and natural proteins corresponding to the NH2-terminal domain. A1 bound to single-stranded DNA-cellulose with higher affinity than the NH2-terminal domain peptides. Protein-induced fluorescence enhancement was used to measure equilibrium binding properties of the proteins. A1 binding to poly (ethenoadenylate) was cooperative with the intrinsic association constant of 1.5 X 10(5) M-1 at 0.4 M NaCl and a cooperativity parameter of 30. The NH2-terminal domain peptides bound noncooperatively and with a much lower association constant. With these peptides and with intact A1, binding was fully reversed by increasing [NaCl]; yet. A1 binding was much less salt-sensitive than binding by the NH2-terminal domain peptides. A synthetic polypeptide analog of the COOH-terminal domain was prepared and was found to bind tightly to poly-(ethenoadenylate). The results are consistent with the idea that the COOH-terminal domain contributes to A1 binding through both cooperative protein-protein interaction and direct interaction with the nucleic acid.

Amino Acids↗