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

Abstract

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.

Association of c‐myc protein with enzymes of DNA replication in high molecular weight fractions from mammalian cells

“…6). Similar results were o 7 3 tained with and other enzymes of DNA replication an B deoxynucleo-C-myc protein can be detected in the replitase preparations from Chinese hamster cells Protein complexes containing DNA olymerase (Y tide biosynthesis have been observed in several laboratories (Ayusawa et al, 1983;Baril et al, 1973;Hammond et al, 1989;Harvey and Pearson, 1988;Reddy 1989;Wickremasinghe et al, 1982). The preparation of one of these complexes, termed the "replitase," has been optimized for Chinese hamster cells (Noguchi 1 2 3 4 5 6 Fig.…”

Replitase: Complete machinery for DNA synthesis

“…Such physical interactions between the enzymes of dNTP synthesis and DNA replication have also been found in mouse FM3A cells (Ayusawa et al, 1983), BHK fibroblasts (Harvey and Pearson, 1988), and human lymphoblasts (Wickremasinghe et al, 1982(Wickremasinghe et al, , 1983. Multienzyme complexes containing the enzymes of both dNTP synthesis and DNA replication were also reported from cells infected with HSV-1 (Sclafani and Fangman, 1984;Harvey and Pearson, 1988) and adenovirus (Yamashita et al, 1977).…”

“…Such physical interactions between the enzymes of dNTP synthesis and DNA replication have also been found in mouse FM3A cells (Ayusawa et al, 1983), BHK fibroblasts (Harvey and Pearson, 1988), and human lymphoblasts (Wickremasinghe et al, 1982(Wickremasinghe et al, , 1983. Multienzyme complexes containing the enzymes of both dNTP synthesis and DNA replication were also reported from cells infected with HSV-1 (Sclafani and Fangman, 1984;Harvey and Pearson, 1988) and adenovirus (Yamashita et al, 1977). As in the case with replication factories (Cardoso et al, 1993) and replicationcompetent multiprotein complexes (Frouin et al, 2002) described above, replitase isolated from HeLa cells also contained cell-cycle regulatory proteins cyclin A and Cdk2 (Jaumot et al, 1994).…”

Nuclear localization of 68 kDa calmodulin‐binding protein is associated with the onset of DNA replication

“…in DNA polymerase and thymidine kinase activities (Table 1). Since DNA polymerase, thymidine kinase, and other enzymes of DNA biosynthesis in the nuclei are known to assemble into multienzyme complex (replitase) responsible for nuclear DNA replication in S phase cells (Reddy and Pardee, 1980;Noguchi et al, 1983;Wickremasinghe et al, 1982;Harvey and Pearson, 1988), we examined if CaM is associated with the replitase fraction isolated from S phase nuclei. As shown in Table 1, similar to DNA polymerase and thymidine kinase, CaM was found to be a n integral component of the complex fraction isolated from the nuclei of S phase, but not of G1 phase cells.…”