DNA Polymerases from Human Cells

Nuclei of HeLa cells contain two separable DNA polymerases which have different properties and cytoplasm contains a single DNA polymerase activity similar to one of the nuclear DNA polymerases. The same distribution of DNA polymerases has been found in the nucleus and cytoplasm of WI-38 cells, a normal diploid human cell line.     

Purification and characterization of varicella-zoster virus-induced DNA polymerase.

Infection of WI-38 human fibroblasts with varicella-zoster virus led to the stimulation of host cell DNA polymerase synthesis and induction of a new virus-specific DNA polymerase. This virus-induced DNA polymerase was partially purified and separated from host cell enzymes by DEAE-cellulose and phosphocellulose column chromatographies. This virus-induced enzyme could be distinguished from host cell enzyme by its chromatographic behavior, template specificity, and its requirement of salt for maximal activity. The enzyme could efficiently use poly(dC).oligo(dG)12-18 as well as poly(dA).oligo(dT)12-18 as template-primers. It required Mg2+ for maximal polymerization activity and was sensitive to phosphonoacetic acid, to which host alpha- and beta-DNA polymerase were relatively resistant. In addition, this induced DNA polymerase activity was enhanced by adding 60 mM (NH4)2SO4 to the reaction mixture.     

Studies on deoxyribonucleic acid polymerase from normal and down’s syndrome skin fibroblasts in vitro

Summary This investigation showed that: (a) hypotonic pH 5.0 extracts of normal and Down’s syndrome fibroblasts cells (DS) exhibit DNase activity which appears to be very similar in nature and extent; (b) DNA polymerase activity was generally increased in DS cells when compared with normal cells; (c) DNA polymerase activity was increased with increased passage age for both normal and DS fibroblasts, but the normal fibroblast was never found to overlap with DS cell activity; and (d) normal cells appear to have a similar DNA polymerase activity at all chronological ages, whereas DS cells exhibit a decrease in activity with increased age of the donor.     

Enhanced Deoxyribonucleic Acid Polymerase Activity in Human Embryonic Kidney Cultures Infected with Adenovirus 2 or 12

Deoxyribonucleic acid (DNA) polymerase activity was induced at approximately 18 to 20 hr after infection of secondary cultures of human embryonic kidney cells with adenovirus type 2 or type 12, and, at 30 to 50 hr after infection, the activity of this enzyme increased two- to threefold. The activity of thymidine kinase was also induced, but the activity of deoxycytidylic deaminase was not. The DNA content per cell at 71 hr after infection was 1.6-fold greater in adenovirus 2-infected cultures, and approximately 2.4-fold greater in adenovirus 12-infected cultures, than in the noninfected cultures. Several properties of DNA polymerase were studied. The enzymes in normal and adenovirus 2- or 12-infected cell extracts were saturated by approximately the same concentration of heat-denatured salmon sperm DNA primer (160 mug/ml); the enzyme activities had a similar broad pH optimum between 7.5 and 9. Extracts prepared from cells infected by either adenovirus did not activate DNA polymerase from noninfected cells, nor did the noninfected cell extracts inhibit enzyme activity of infected cell extracts. DNA polymerase in both normal and adenovirus 2- or 12-infected cells was located predominantly in the nucleus. In each case, the cytoplasm had only 30% of the enzyme activity of the nucleus. At 40 hr after infection with adenovirus 2 or 12, the activities of the enzyme in the nuclear and cytoplasmic fractions increased two- to threefold. Puromycin, an inhibitor of protein synthesis, prevented DNA polymerase induction when added to cultures during the 18- to 30-hr postinfection period, and it arrested the additional increase in enzyme activity when added after enzyme induction began. However, the increases in both DNA polymerase and thymidine kinase activities took place after treatment of infected cultures with 1-beta-d-arabinofuranosylcytosine, an inhibitor of DNA synthesis and adenovirus growth.     

DNA polymerase alpha-DNA primase from human placenta. Immunoaffinity purification and preliminary characterization

Highly purified DNA polymerase alpha-DNA primase from normal human tissue (human placenta) has been prepared by immunoaffinity purification on immobilized anti-human DNA polymerase alpha monoclonal antibody SJK 287-38. According to data from SDS electrophoresis this preparation consists of subunits of 180, 160, 145, 140 kDa (a cluster of DNA-polymerizing subunits), 73 kDa (function unknown) and 59, 52 kDa (corresponding to primase). Three active enzyme forms of 270, 460 and 575 kDa have been revealed using native electrophoresis followed by detection of DNA polymerase activity.     

Effect of glycerol and low pH on heat-induced cell killing and loss of cellular DNA polymerase activities in Chinese hamster ovary cells

A whole-cell assay technique for DNA polymerase alpha and beta was used to measure the activities of both enzymes in Chinese hamster ovary (CHO) cells after hyperthermic treatment of 42.2 - 45.5 degrees C in acidic or basic environment and in the presence or absence of 5% glycerol. Cell survival was measured at the same time, and the DNA polymerase activities were correlated with survival. The results show a positive correlation between cell killing by heat and loss of DNA polymerase beta activity, both when cells were sensitized to heat by treatment at pH 6.7 with or without glycerol and when cells were protected from heat by treatment with 5% glycerol at pH 7.4 or 6.7. The results show a poor correlation between loss of DNA polymerase alpha activity and cell survival; i.e., compared to cell killing, the loss of DNA polymerase alpha activity was sensitized to heat more by acidic treatment without glycerol and was protected less from heat by glycerol treatment at normal physiological pH (pH 7.4). However, cell killing and loss of polymerase alpha activity did correlate well for sensitization to heat by acidic treatment in the presence of glycerol and for protection from heat by glycerol treatment at low pH. These results considered with other hyperthermia-polymerase studies suggest that heat effects on membranes can apparently result in changes in environmental conditions within the cell (secondary effects), which can in turn alter polymerase activities and/or the direct or secondary effect of heat on the polymerase enzymes. Furthermore, loss of polymerase beta activity serves as a better index of thermal damage resulting in cell death than loss of alpha activity.     

Intranuclear dynamics of DNA polymerase α differs between the transplanted R3230AC mammary adenocarcinomas and the host mammary gland depending on lactation cycle

DNA polymerase α activity was markedly higher in all nuclear subfractions, including nuclear matrix, from transplanted R3230AC mammary adenocarcinomas than in the analogous fractions from mammary gland of same tumor-bearing pregnant or lactating rats. Changes in host lactational status had no significant effect on subnuclear distribution of tumor DNA polymerase α activity, with the majority (60–75%) localized in soluble nucleoplasm and a significant amount (13–20%) retained in the nuclear matrix. In the host mammary gland, nuclear matrix-bound DNA polymerase α was highest, accounting for 48% of total nuclear activity, during late pregnancy when mammary cells undergo rapid raplication. During lactation, when cells in mammary gland cease to divide, only 8% of enzyme activity was in the nuclear matrix, while the majority (60–80%) of DNA polymerase α activity was localized in nucleoplasm. In both R3230AC tumor and mammary gland regardless of host's lactational status, the majority (60–80%) of DNA polymerase β activity was localized in the high salt-soluble chromatin. These present data thus suggest that, regardless of host lactational status, R3230AC tumor has many cycling cells, each with a large pool of DNA polymerase α molecules maintaining maximal and constant replicative activity, while normal mammary gland cells have a smaller pool of DNA polymerase α which become primarily matrix-bound only during active cell replication during late pregnancy. A constant localization of nuclear DNA polymerase β in chromatin in both mammary gland and the tumor suggest it is not important in mammary cell proliferation.     

Intranuclear dynamics of DNA polymerase alpha differs between the transplanted R3230AC mammary adenocarcinomas and the host mammary gland depending on lactation cycle

DNA polymerase alpha activity was markedly higher in all nuclear subfractions, including nuclear matrix, from transplanted R3230AC mammary adenocarcinomas than in the analogous fractions from mammary gland of same tumor-bearing pregnant or lactating rats. Changes in host lactational status had no significant effect on subnuclear distribution of tumor DNA polymerase alpha activity, with the majority (60-75%) localized in soluble nucleoplasm and a significant amount (13-20%) retained in the nuclear matrix. In the host mammary gland, nuclear matrix-bound DNA polymerase alpha was highest, accounting for 48% of total nuclear activity, during late pregnancy when mammary cells undergo rapid raplication. During lactation, when cells in mammary gland cease to divide, only 8% of enzyme activity was in the nuclear matrix, while the majority (60-80%) of DNA polymerase alpha activity was localized in nucleoplasm. In both R3230AC tumor and mammary gland regardless of host's lactational status, the majority (60-80%) of DNA polymerase beta activity was localized in the high salt-soluble chromatin. These present data thus suggest that, regardless of host lactational status, R3230AC tumor has many cycling cells, each with a large pool of DNA polymerase alpha molecules maintaining maximal and constant replicative activity, while normal mammary gland cells have a smaller pool of DNA polymerase alpha which become primarily matrix-bound only during active cell replication during late pregnancy. A constant localization of nuclear DNA polymerase beta in chromatin in both mammary gland and the tumor suggest it is not important in mammary cell proliferation.     

Repair of benzo[a]pyrene-initiated DNA damage in human cells requires activation of DNA polymerase alpha

Normal human fibroblasts treated with r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) yielded DNA polymerase alpha with elevated levels of activity, incorporated [3H]thymidine as a function of unscheduled DNA synthesis, and exhibited restoration of normal DNA-strand length as a function of unscheduled DNA synthesis. Lipoprotein-deficient fibroblasts treated with BPDE did not show elevated levels of DNA polymerase alpha activity, exhibited minimal [3H]thymidine incorporation, and had fragmented DNA after 24 h of repair in the absence of lipoprotein or phosphatidylinositol supplementation. When DNA polymerase beta activity was inhibited, cells with normal lipoprotein uptake exhibited [3H]thymidine incorporation into BPDE-damaged DNA but did not show an increase in DNA-strand length. DNA polymerase alpha activity and [3H]thymidine incorporation in lipoprotein-deficient fibroblasts increased to normal levels when the cells were permeabilized and low-density lipoproteins or phosphatidylinositol were introduced into the cells. DNA polymerase alpha isolated from normal human fibroblasts, but not from lipoprotein-deficient fibroblasts, showed increased specific activity after the cells were treated with BPDE. When BPDE-treated lipoprotein-deficient fibroblasts were permeabilized and 32P-ATP was introduced into the cells along with lipoproteins, 32P-labeled DNA polymerase alpha with significantly increased specific activity was isolated from the cells. These data suggest that treatment of human fibroblasts with BPDE initiates unscheduled DNA synthesis, as a function of DNA excision repair, which is correlated with increased activity of DNA polymerase alpha, and that increased DNA polymerase alpha activity may be correlated with phosphorylation of the enzyme in a reaction that is stimulated by low-density lipoprotein or by the lipoprotein component, phosphatidylinositol.     

Selective Modification of Adenovirus Replication Can Be Achieved through Rational Mutagenesis of the Adenovirus Type 5 DNA Polymerase

Mutations that reduce the efficiency of deoxynucleoside (dN) triphosphate (dNTP) substrate utilization by the HIV-1 DNA polymerase prevent viral replication in resting cells, which contain low dNTP concentrations, but not in rapidly dividing cells such as cancer cells, which contain high levels of dNTPs. We therefore tested whether mutations in regions of the adenovirus type 5 (Ad5) DNA polymerase that interact with the dNTP substrate or DNA template could alter virus replication. The majority of the mutations created, including conservative substitutions, were incompatible with virus replication. Five replication-competent mutants were recovered from 293 cells, but four of these mutants failed to replicate in A549 lung carcinoma cells and Wi38 normal lung cells. Purified polymerase proteins from these viruses exhibited only a 2- to 4-fold reduction in their dNTP utilization efficiency but nonetheless could not be rescued, even when intracellular dNTP concentrations were artificially raised by the addition of exogenous dNs to virus-infected A549 cells. The fifth mutation (I664V) reduced biochemical dNTP utilization by the viral polymerase by 2.5-fold. The corresponding virus replicated to wild-type levels in three different cancer cell lines but was significantly impaired in all normal cell lines in which it was tested. Efficient replication and virus-mediated cell killing were rescued by the addition of exogenous dNs to normal lung fibroblasts (MRC5 cells), confirming the dNTP-dependent nature of the polymerase defect. Collectively, these data provide proof-of-concept support for the notion that conditionally replicating, tumor-selective adenovirus vectors can be created by modifying the efficiency with which the viral DNA polymerase utilizes dNTP substrates.     

Human cytomegalovirus. III. Virus-induced DNA polymerase.

Infection of WI-38 human fibroblasts with human cytomegalovirus (CMV) led to the stimulation of host cell DNA polymerase synthesis and induction of a novel virus-specific DNA polymerase. This cytomegalovirus-induced DNA polymerase was purified and separated from host cell enzymes by DEAE-cellulose and phosphocellulose column chromatographies. It can be distinguished from host cell enzymes by chromatographic behavior, template primer specificity, sedimentation property, and the requirement of salt for maximal activity. This virus-induced enzyme has a sedimentation coefficient of 9.2S and is found in both the nuclei and cytoplasm of virus-infected cells, but not in uninfected cells. This enzyme could efficiently use activated calf-thymus DNA, oly(dA)-oligo(dT)12-18, and poly(dC)-oligo(dG)12-18 as template primers, especially poly(dA)-oligo(dT)12-18, but it could not use poly(rA)-oligo(dT)12-18, poly(rC)-oligo(dG)12-18, or oligo(dT)12-18. The enzyme requires Mg2+ for maximal activity, is sensitive to p-hydroxymercuribenzoate, and is not a zinc metalloenzyme. In addition, the cytomegalovirus-induced DNA polymerase activity can be enhanced by adding 0.06 to 0.12 M NaCl or 0.03 to 0.06 M (NH4)2SO4 to the reaction mixture.     

PDIP38 is a novel mitotic spindle-associated protein that affects spindle organization and chromosome segregation

In order to maintain genomic integrity during mitosis, cells assemble the mitotic spindle to separate sister chromosomes to the two daughter cells. A variety of motor- and non motor-proteins are involved in the organization and regulation of this complex apparatus. DNA polymerase δ-interacting protein 38 (PDIP38) is a highly conserved protein and has so far been shown to be a cytoplasmic and nuclear protein. Cell cycle dependent nuclear localization and the interaction with DNA polymerase δ and proliferating cell nuclear antigen (PCNA) indicate a role for PDIP38 in DNA modification and/or proliferation. Here, we show for the first time that PDIP38 localizes to the mitotic spindle throughout mitosis. Using anti-PDIP38 antibody injections and siRNA silencing, we demonstrate that PDIP38 loss-of-function causes problems with spindle organization, aberrant chromosome segregation, and multinucleated cells. Taken together, the data indicate different roles for PDIP38 in safeguarding a proper cell division at various stages of the cell cycle, including DNA synthesis and repair, organization of the mitotic spindle and chromosome segregation.     

Polyoma virus minichromosomes: associated enzyme activities.

Polyoma minichromosomes were isolated and fractionated on glycerol gradients as described by Gourlie et al. (J. Virol. 38:805-814, 1981). Specific assays for DNa polymerases alpha, beta, and gamma, DNA topoisomerase I, and RNase H were carried out on each fraction. The number of units of activity in each fraction was compared with the number of total polyoma and replicative intermediate DNA molecules in each fraction determined by quantitative electron microscopy (M. R. Krauss and R. M. Benbow, J. Virol. 38:815-825, 1981). DNA polymerase alpha cosedimented with polyoma replicative intermediate DNA molecules. DNA polymerase beta and DNA topoisomerase I activities sedimented with mature polyoma minichromosomes. Although the bulk of RNase H activity sedimented in the minichromosome region, the peak of activity was found one fraction behind the peak of mature minichromosomes. Virtually no DNA polymerase gamma activity cosedimented with polyoma minichromosomes.     

Identification of a beta-like DNA polymerase activity in bovine heart mitochondria

A new DNA polymerase activity, distinct from DNA polymerase gamma, has been identified in bovine heart mitochondria. First detected among proteins isolated in a complex with mitochondrial DNA, the DNA polymerase activity has been partially purified 47,000-fold. Enzyme activity separates from DNA polymerase gamma on several chromatographic columns and appears to copurify with a 38 +/- 2-kDa polypeptide. Unlike DNA polymerase gamma, this enzyme is relatively resistant to inhibition by N-ethylmaleimide and dideoxynucleotides, has moderately low monovalent and high divalent cation requirements, and possesses 20-fold-higher apparent K(m) values for deoxynucleotides. The enzyme polymerizes deoxynucleotides onto a primed template DNA in a relatively nonprocessive fashion and lacks a detectable 3' to 5' exonuclease activity. Many of these characteristics resemble a beta-like mitochondrial DNA polymerase previously identified in, and considered unique to, trypanosomes. We propose that the bovine and trypanosomal enzymes are related and represent a new class of ubiquitous mitochondrial DNA polymerases.     

Nuclear deoxyribonucleic acid polymerases of liver.

Hepatic nuclei that are isolated in aquenous solutions of low ionic strength or glycerol contain all or nearly all the nonmitochondrial DNA polymerase activity of the cell. The presence of polymerase activity in the cytoplasm is due to extraction of nuclear enzymes by buffer and inorganic salts. Even with low ionic strength solutions, some leaching of nuclear enzymes occurs if the concentration of liver in the homogenizing medium is greater than 10%. As defined by sucrose gradient analysis, the normal adult rat liver nucleus contains mainly or entirely a single species of DNA polymerase (3.2 S) whereas the regenerating nucleus after 70% hepatectomy has an additional enzyme (7.1 S). The total activity of regenerating nuclei is about twice the normal value. The increase resides in the 7.1 S activity. The 7.1 S DNA polymerase had been purified partially from regenerating liver nuclei (isolated in low ionic strength solutions) and cytosol (prepared under conditions of nuclear enzyme extraction). The properties of the activity from the two sources are indistinguishable. A mixture of albumin and spermidine enhances by several-fold the activities of the 3.2 S and 7.1 S DNA polymerases. In the presence of spermidine, but not in its absence, the activity of the 7.1 S DNA polymerase is strictly proportional to the amount of the enzyme preparation.     

Temperature-Sensitive Mutants for the Replication of Plasmids in Escherichia coli: Requirement for Deoxyribonucleic Acid Polymerase I in the Replication of the Plasmid ColE1

An Escherichia coli mutant (polA1), defective in deoxyribonucleic acid (DNA) polymerase I, (EC 2.7.7.7) is unable to maintain colicinogenic factor E1 (ColE1), whereas several sex factor plasmids are maintained normally in this strain. polA1 mutant strains containing these sex factor plasmids do not exhibit a readily detectable plasmid-induced polymerase activity. A series of E. coli mutants that are temperature sensitive for ColE1 maintenance, but able to maintain other plasmids, were isolated and shown to fall into two phenotypic groups. Mutants in one group are defective specifically in ColE1 maintenance at 43 C, but exhibit normal DNA polymerase I activity. Mutations in the second group map in the polA gene of E. coli, and bacteria carrying these mutations are sensitive to methylmethanesulfonate (MMS). Revertants that were selected either for MMS resistance or the ability to maintain ColE1 were normal for both properties. The DNA polymerase I enzyme of two of these mutants shows a pronounced temperature sensitivity when compared to the wild-type enzyme. An examination of the role of DNA polymerase I in ColE1 maintenance indicates that it is essential for normal replication of the plasmid. In addition, the presence of a functional DNA polymerase I in both the donor and recipient cell is required for the ColV-promoted conjugal transfer of ColE1 and establishment of the plasmid in the recipient cell.     

O6-methylguanine methyltransferase increases before S phase in normal human cells but does not increase in hypermutable Bloom's syndrome cells

The regulation of the O6-methylguanine methyltransferase was examined during cell proliferation in hypermutable Bloom's syndrome fibroblasts and normal human skin fibroblasts. During synchronous growth following serum stimulation normal human cells enhanced methyltransferase activity 2.4-fold in the absence of exogenous damage as a normal regulatory event during the cell cycle. Methyltransferase activity was increased prior to the induction of DNA replication and of DNA polymerase and was diminished when each replicative activity was maximal. In contrast, although methyltransferase levels in quiescent cells are equivalent, hypermutable Bloom's syndrome cells did not increase methyltransferase at any interval in the cell cycle.     

Human DNA polymerase alpha gene expression is cell proliferation dependent and its primary structure is similar to both prokaryotic and eukaryotic replicative DNA polymerases.

We have isolated cDNA clones encoding the human DNA polymerase alpha catalytic polypeptide. Studies of the human DNA polymerase alpha steady-state mRNA levels in quiescent cells stimulated to proliferate, or normal cells compared to transformed cells, demonstrate that the polymerase alpha mRNA, like its enzymatic activity and de novo protein synthesis, positively correlates with cell proliferation and transformation. Analysis of the deduced 1462-amino-acid sequence reveals six regions of striking similarity to yeast DNA polymerase I and DNA polymerases of bacteriophages T4 and phi 29, herpes family viruses, vaccinia virus and adenovirus. Three of these conserved regions appear to comprise the functional active site required for deoxynucleotide interaction. Two putative DNA interacting domains are also identified.     

Reduction of DNA primase activity in aging but still proliferating cells

The basis of the well-known decline in cell proliferation with increasing passage number of human diploid fibroblast-like cell cultures is not known. It has been found that DNA synthesis was deficient in the remaining but still proliferating cells, but when appropriate corrections reflecting the remaining dividing cells were made, the amount of DNA polymerase alpha bound to nuclear matrices was normal [Collins and Chu: Journal of Cellular Physiology 124:165-173, 1985]. In the present study, the declining percentages of S-phase and dividing cells were determined to provide better estimates of functional culture age than passage number. The amounts of DNA polymerase alpha and DNA primase activity were determined in cell lysates, permeabilized cells, and bound to nucleoids, which are residual nuclear structures similar to nuclear matrices except that no DNase-digestion step is employed. As expected, IMR 90 DNA synthesis declined with age, even after corrections for the declining numbers of proliferating cells. DNA polymerase alpha and DNA primase activity in cell lysates, permeabilized cells, and bound to nucleoids declined with increasing age. However, after appropriate corrections for the declining fraction of proliferating cells, the only activity that declined was that of DNA primase bound to nucleoids. Thus, a decrease in the binding of DNA primase to the nuclear site of DNA synthesis may account for the decreased DNA synthesis in aging but still proliferating cells.