DNA-binding protein associated with herpes simplex virus DNA polymerase

Vaughan, Patrick; Purifoy, Dorothy J. M.; Powell, Kenneth L.

doi:10.1128/jvi.53.2.501-508.1985

Cited by 81 publications

(60 citation statements)

References 27 publications

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“…It is possible that only a loose association of the DNA polymerase with RR and TK exists, or that the polymerase is associated with other enzymes and proteins that were not assayed. Other reports have shown, for example, the copurification of both DNA-binding proteins and topoisomerase with HSV-1-induced DNA polymerase (Biswal et al, 1983;Vaughan et al, 1985). Thus, in the virus-infected cell the production and utilisation of DNA precursors might not be tightly coupled or may not even be coupled at all, a possibility discussed above also for the uninfected cell.…”

Section: Loss Of Protein From Permeabilized Cells Was Estimatedmentioning

confidence: 97%

Search for multienzyme complexes of DNA precursor pathways in uninfected mammalian cells and in cells infected with herpes simplex virus type I

Harvey

Pearson

1988

Journal Cellular Physiology

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Confirmatory evidence for the existence of a multienzyme complex of DNA precursor pathways in mammalian cells was obtained. Using neutral sucrose gradient centrifugation of cell lysates we found that at least five enzymes involved in DNA precursor metabolism in uninfected. S-phase BHK-cell fibroblasts cosediment at a common rate, indicative of a multienzyme complex. The enzymes include DNA polymerase thymidine kinase, ribonucleotide reductase, dihydrofolate reductase, and NDP-kinase. This complex was partially, but not completely, disrupted when lysates from GO-phase cells were centrifuged. Using lysates from cells infected with herpes simplex virus (HSV) type I some of the virus-induced ribonucleotide reductase and a minor proportion of the HSV-thymidine kinase cosedimented rapidly. The virus-induced DNA polymerase sedimented independently near the middle of the gradient, in contrast to the behaviour of the host polymerase. The enzyme associations observed were disrupted by NaCl or by inclusion of ethylenediamine tetraacetic acid during the cell lysis procedure, instead of the usual EGTA. These results indicate the importance of ionic forces in maintaining the enzyme complexes. The bulk of the DNA and the RNA present in the lysates did not sediment at the same rate as the complexes, showing that the enzymes were not simply adhering nonspecifically to these polyanions. Newly synthesised radiolabeled DNA (15 min pulse with [3H]thymidine) was not preferentially associated with the enzymes, but some functional DNA was evident in the enzyme complex fraction from the uninfected S-phase cells. DNA polymerase activity in this fraction did not require, nor was it stimulated by, exogenous "activated" DNA. Added DNA primer-template was required, however, for maximal activity of the polymerase in gradient fractions derived from GO-phase cells and from HSV-infected cells. No evidence for channeling of ribonucleotide precursors into DNA of permeabilized cells (uninfected or HSV-infected) was detected. Most rCDP was incorporated into RNA. In the uninfected, S-phase cells about 10 pmol/10(6) cells/90 min of rCDP residues was incorporated into DNA compared with 120 pmol/10(6) cells/90 min when radiolabeled dCTP was used. Nonradioactive dCTP present in equimolar concentration in the incubation with labeled rCDP did not, however, diminish the incorporation of label from the ribonucleotide. In permeabilized HSV-infected cells incorporation of radiolabel from rCDP into DNA was barely detectable.

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Section: Loss Of Protein From Permeabilized Cells Was Estimatedmentioning

confidence: 97%

Search for multienzyme complexes of DNA precursor pathways in uninfected mammalian cells and in cells infected with herpes simplex virus type I

Harvey

Pearson

1988

Journal Cellular Physiology

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“…The product of the UL42 gene (UL42) is one of seven proteins, required to replicate plasmids containing an HSV origin of replication (Wu et al, 1988), and is essential for viral DNA replication in vivo (Marchetti et al, 1988;Schaffer et al, 1973). Initially, UL42 was characterized as a phosphorylated 65-kDa double-stranded (ds)DNA binding protein being associated with purified HSV pol from serotype 1-and 2-infected cells (Gallo et al, 1988(Gallo et al, , 1989Marsden et al, 1987;Powell and Purifoy, 1977;Vaughan et al, 1985). From the open reading frame of the UL42 gene of HSV-1 strain 17, only a molecular weight of 51, 156 can be predicted .…”

Section: Introductionmentioning

confidence: 99%

DNA and Protein Interactions of the Small Subunit of Herpes Simplex Virus Type 1 DNA Polymerase

1999

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Herpes simplex virus DNA polymerase (HSV pol) holoenzyme consists of a large catalytic (UL30 gene product) and a small auxiliary subunit (UL42 gene product). The DNA binding of HSV pol, its cofactor, and the assembled holoenzyme complex was studied by bandshift analysis using purified proteins expressed via recombinant baculovirus. The functional activity of the recombinant UL42, purified by phenyl-Sepharose chromatography, was confirmed by its ability (1) to convert the salt sensitivity of both, 3'-5' exonuclease and polymerase, activities of HSV pol and (2) to enhance the processivity of polymerization. Bandshift analyses revealed that the HSV pol holoenzyme-DNA complex was stably formed up to a salt concentration of 50 mM ammonium sulfate, indicating that the restricted DNA and protein interactions of both HSV pol and UL42 are responsible for the observed salt preference of the HSV pol holoenzyme under standard assay conditions in vitro. Studies of the assembly of the holoenzyme complex demonstrated that initial DNA binding of HSV pol was advantageous for the formation of the HSV pol holoenzyme-DNA complex.

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“…Coen, manuscript submitted for publication), that prevent the isolation of Pol in 'substrate' quantities (9). Additionally, it has been difficult to purify Pol away from other cellular and viral proteins, especially the HSV-encoded double stranded DNA binding protein, which is a product of the UL42 gene (10)(11)(12)(13)(14). This has made it difficult to determine whether activities associated with HSV Pol are intrinsic to the polypeptide product of the HSV pol gene (UL30) or, if they are intrinsic, whether they require other proteins for stimulation.…”

Section: Introductionmentioning

confidence: 99%

Enzymatic activities of overexpressed herpes simplex virus DNA polymerase purified from recombinant baculovirus-infected insect cells

Marcy¹,

Olivo

Challberg

et al. 1990

Nucl Acids Res

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Biochemical characterization of the herpes simplex virus (HSV) DNA polymerase, a model DNA polymerase and an important target for antiviral drugs, has been limited by a lack of pure enzyme in sufficient quantity. To overcome this limitation, the HSV DNA polymerase gene was introduced into the baculovirus, Autographa californica nuclear polyhedrosis virus, under the control of the polyhedrin promoter to give rise to a recombinant baculovirus, BP58. BP58-infected Spodoptera frugiperda insect cells expressed a polypeptide that was indistinguishable from authentic polymerase by several immunological and biochemical properties, at levels approximately ten-fold higher per infected cell than found in HSV-infected Vero cells. The DNA polymerase was purified to apparent homogeneity from BP58-infected insect cells. Using activated DNA as primer-template, the purified enzyme exhibited specific activity similar to that of enzyme isolated from HSV-infected Vero cells, indicating that additional polymerase-associated proteins from HSV-infected cells are not critical for activity with this primer-template. 3'-5' exonuclease activity co-purified with the BP58-expressed HSV DNA polymerase, demonstrating that this activity is intrinsic to the polymerase polypeptide. The purified enzyme also exhibited RNAse H activity. The recombinant baculovirus should permit detailed biochemical and biophysical studies of this enzyme.

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DNA-binding protein associated with herpes simplex virus DNA polymerase

Cited by 81 publications

References 27 publications

Search for multienzyme complexes of DNA precursor pathways in uninfected mammalian cells and in cells infected with herpes simplex virus type I

Search for multienzyme complexes of DNA precursor pathways in uninfected mammalian cells and in cells infected with herpes simplex virus type I

DNA and Protein Interactions of the Small Subunit of Herpes Simplex Virus Type 1 DNA Polymerase

Enzymatic activities of overexpressed herpes simplex virus DNA polymerase purified from recombinant baculovirus-infected insect cells

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