Evidences for the function of DNA polymerase-beta in unscheduled DNA synthesis.

The activities of DNA polymerase-alpha and -beta isolated from pig spleen were determined at different temperatures and in the presence of different concentrations of inhibitors. The results were compared with parallel estimations of replicative DNA synthesis and UV-induced repair synthesis in spleen cells. In respect to pCMB and aCTP, polymerase-alpha is more sensitive than polymerase-beta and similarly is replication more sensitive than repair. Repair synthesis and the activity of polymerase-beta decreases at temperatures higher than 40 degrees C whereas both replication and the activity of polymerase-alpha are greatly stimulated at elevated temperatures with optima of 45 degrees C (polymerase-alpha) and 41 degrees C (replication). The results favour the hypothesis that polymerase-beta is involved in repair synthesis.     

DNA polymerase activity in a repair-deficient human cell line

A human low-density-lipoprotein (LDL) receptor-deficient diploid fibroblast cell line (GM1915) was determined to be short patch competent (DNA polymerase-beta) and long patch deficient (DNA polymerase-alpha) for DNA excision repair. Analysis of DNA from GM1915 cells or from WI38 control cells, following treatment with a mutagen known to initiate long patch excision repair, showed that GM1915 cells exhibited decreased resynthesis of oligonucleotide segments excised during repair. When cells deficient in DNA polymerase-alpha activity were permeabilized to permit LDL entry, repair synthesis immediately increased. These data suggest that DNA polymerase-alpha is not activated by mutagen treatment in GM1915 cells and that introduction of LDL into the cells results in activation of the enzyme.     

Delayed and reduced cell replication and diminishing levels of DNA polymerase-alpha in regenerating liver of aging mice

Kinetics of cell replication was compared in regenerating livers of Mus musculus at ages ranging between 6 and 32 months. Incorporation of [3H]-thymidine into DNA and autoradiographic analysis showed that the maximal extent of DNA replication following partial hepatectomy became delayed with age. Furthermore, the total fraction of parenchymal and nonparenchymal cells in S phase at different intervals during regeneration diminished as mice aged. The specific activity of DNA polymerase-alpha, the putative replicative enzyme, declined progressively during aging. The specific activity of DNA polymerase-beta, the purported repair enzyme, declined to an appreciably lesser extent during the lifespan of the mouse. No evidence was found for the appearance of a specific inhibitor of polymerase-alpha in senescent mouse liver. Also, the bulk of the activities of both hepatic DNA polymerase-alpha and -beta remained localized in the cell nucleus throughout the lifetime of the animal and were mainly associated with chromatin.     

Immunological reagents for comparisons of DNA polymerase-alpha and DNA polymerase-beta

Antibodies to homogeneous calf thymus DNA polymerase-beta and calf thymus DNA polymerase-alpha preparations were raised in rabbits. The antiserum against calf thymus DNA polymerase-beta cross-reacts with all vertebrate DNA polymerase-beta preparations tested, but does not cross-react with trypanosome DNA polymerase-beta, DNA polymerase-gamma, terminal transferase, yeast DNA polymerases, and Escherichia coli DNA polymerase I. The antibodies against calf thymus DNA polymerase-alpha cross-react with DNA polymerase-alpha from mouse, human, and chicken, but do not cross-react with DNA polymerase-alpha from sea urchin embryos and Drosophila embryos, DNA polymerase-beta, DNA polymerase-gamma, terminal transferase, yeast DNA polymerases, and E. coli DNA polymerase I.     

Inhibition of DNA polymerase-alpha and -beta of calf thymus by 1-beta-D-arabinofuranosylcytosine-5'-triphosphate.

1-beta-D-Arabinofuranosylcytosine 5'-triphosphate (araCTP), an active form of a inhibitor of DNA replication, 1-beta-D-arabinofuranosylcytosine (araC) was tested for its inhibitory action on the DNA polymerase-alpha and -beta (EC 2.7.7.7) purified from calf thymus. The reaction of DNA polymerase-alpha was shown to be more sensitive to the inhibition by araCTP than that of DNA polymerase-beta. The mode of the inhibition by araCTP was competitive to dCTP in the reaction catalysed by either DNA polymerase-alpha or -beta. The Ki value of DNA polymerase-beta for araCTP was 32 micron; eight times higher than that of DNA polymerase-alpha (4 micron) for this inhibition.     

The mode of inhibitory action by pyridoxal 5-phosphate on DNA polymerase-alpha and -beta.

The kinetics of the inhibition of DNA polymerases-alpha and -beta from sea urchin embryos by pyridoxal 5-phosphate were studied. The inhibition of DNA polymerase-alpha activity by pyridoxal 5-phosphate was competitive with activated DNA but noncompetitive with each deoxynucleoside triphosphate. With poly(dC)-oligo(dG)12-18 as a template-primer, however, the inhibition of DNA polymerase-alpha was competitive with dGTP but noncompetitive with the template-primer. These results suggest that DNA polymerase-alpha interacts with activated DNA and poly(dC)-oligo(dG)12-18 in different ways. The inhibition of DNA polymerase-beta by pyridoxal 5-phosphate was competitive with deoxynucleoside triphosphate using activated DNA as a template-primer and noncompetitive with activated DNA. Using poly(rA)-oligo(dT)12-18 as a template-primer, DNA polymerase-beta activity yielded sigmoid curves against both dTTP and the template-primer concentrations and was inhibited by pyridoxal 5-phosphate noncompetitively with respect to both dTTP and the template-primer. These results indicate that the inhibitory mode of DNA polymerase-alpha by pyridoxal 5-phosphate is different from that of DNA polymerase-beta.     

Regenerating rat liver DNA polymerases: disimilitude or relationship between nuclear and cytoplasmic enzymes?

The possible relationship between the nuclear and cytoplasmic DNA polymerases of regenerating rat liver was studied by sucrose gradient analysis, salt dissociation, and with specific inhibitors. After aqueous subcellular fractionation and removal of the nuclear membranes, three species of DNA-dependent DNA polymerases were characterized: 1) a DNA polymerase-beta in the nuclei. 2) a DNA polymerase-alpha in the cytosol which was not dissociated at high salt concentrations; and 3) an intermediate form in the cytosol and in the Triton wash containing the nuclear membranes. The latter form behaved like DNA polymerase-alpha et low salt concentration but was dissociated at high salt concentrations to a low molecular weight species with properties like DNA polymerase-beta (resistance to inhibition by N-ethylmaleimide, heparin and KCL). In vitro reassociation experiments suggest that this intermediate form corresponds to the association of DNA polymerase-beta with a membrane component or cytoplasmic protein(s) which appear(s) in regenerating rat liver.     

Inhibition of polymerases-alpha and -beta completely blocks DNA repair induced by UV irradiation in cultured mouse neuronal cells

The effects of hydroxyurea, aphidicolin and dideoxythymidine on UV-induced DNA repair of mouse neuronal granular cells were studied. Aphidicolin, which is considered a specific inhibitor of polymerase-alpha, decreased spontaneous DNA synthesis by 93% and totally suppressed DNA repair. Dideoxythymidine, an inhibitor of polymerase-beta, was more potent in decreasing scheduled DNA synthesis than aphidicolin, and also completely blocked the UV-induced DNA repair. Hydroxyurea, a specific inhibitor of ribonucleotide reductase, inhibited scheduled DNA synthesis, but unscheduled DNA synthesis after UV irradiation was always well detectable. Our data suggest that in neuronal cells from 5 to 10 days old mice both polymerases-alpha and -beta are required for both DNA synthesis and repair. These two enzymes may act jointly in filling up the gaps along the DNA molecule and elongating the DNA chain.     

Studies on the purification and properties of a 6.8-S DNA polymerase activity found in calf-thymus DNA polymerase-alpha fraction.

The heterogeneity of calf thymus DNA polymerase-alpha has been further investigated. In particular, an enzyme (enzyme D) which exhibits higher activity on poly(dA) . (dT)10 (A:T = 20:1) compared with that on activated DNA, has been further purified and its properties compared with two other activities of the DNA polymerase-alpha fraction (enzymes A1 and C) which do not show a preference for poly(dA) . (dT)10 over activated DNA. As with A1 and C, enzyme D was shown to have many of the characteristic properties of DNA polymerase-alpha in that it is an acidic protein as judged by its binding to DEAE-cellulose, has a molecular weight of about 140000, does not use a poly (A) . (dT)10 template-initiator complex and is inhibited by N-ethylmaleimide. It exhibits anomalous gel filtration behaviour on Sepharose 6B and it binds relatively weakly to DNA-cellulose compared with DNA polymerase-beta. The extreme sensitivity of enzyme D to inhibtion by N-ethylmaleimide distinguishes it from A1 and C, as does its elution position from a DEAE-cellulose column. On the other hand enzymes C and D are readily inactivated by heating at 45 degrees C unlike enzyme A1. The possible interrelationships of the multiple activities of calf thymus DNA polymerase-alpha are discussed.     

Variation of DNA polymerases-alpha, -beta. and -gamma during perinatal tissue growth and differentiation.

The activities of the three known DNA polymerases-alpha, beta-, and -gamma were determined in rat brain neurons, cardiac muscle and spleen, and were correlated with the rate of cell proliferation during perinatal development. In neurons and cardiac muscle, which stop dividing before birth, DNA polymerase-alpha activity drops sharply from a high level with the approach of term and disappears at approximately two weeks postnatal age. In contrast, alpha-polymerase activity is almost absent in spleen during late gestation, when the rate of cell division is low, and increases abruptly after birth with the sudden onset of cell proliferation. These data give further evidence for an involvement of DNA polymerase-alpha in DNA replication. DNA polymerase-beta and -gamma activities show essentially no correlation with the rate of cell division. Thus, these enzymes are probably responsible for repair type processes rather than for DNA replecation.     

Effect of drugs on deoxyribonucleic acid synthesis in isolated mammalian cell nuclei. Comparison with partially purified deoxyribonucleic acid polymerases.

In order to ascertain the identity of the DNA-dependent DNA polymerase responsible for the observed DNA synthesis in nuclei isolated from baby-hamster kidney (BHK-21/C13) cells a comparative study was carried out on the effects of some drugs, reported to influence DNA synthesis, on DNA synthesis catalysed by these nuclei and by partially purified DNA polymerase-alpha and -beta. In all cases DNA synthesis by isolated nuclei and polymerase-alpha was inhibited to similar extents by N-ethylmaleimide, p-hydroxymercuribenzoate, novobiocin, heparin and phosphonoacetic acid; polymerase-beta was much less affected by these compounds. Ethidium bromide inhibited all DNA synthesis to similar extents, although at low concentrations (about 2 microgram/ml) synthesis in isolated nuclei was stimulated. The results are discussed in relation to the proposal that DNA polymerase-alpha catalyses the covalent extension of Okazaki fragments that these nuclei carry out in vitro.     

Dependence of cell survival on DNA repair in human mononuclear phagocytes.

Mononuclear phagocytes play a central role in the pathogenesis of chronic inflammatory diseases. It is therefore important to define chemotherapeutically exploitable metabolic pathways that distinguish monocytes from other cell types. Blood monocytes do not synthesize deoxynucleotides de novo, and their transformation to macrophages occurs without cell division. Whether or not monocytes can repair DNA damage, and whether or not DNA repair is necessary for their survival, is unknown. The present experiments demonstrate that normal human monocytes, unlike neutrophils, rapidly repair DNA strand breaks induced by gamma-irradiation. Monocyte extracts contain functional immunoreactive DNA polymerase-alpha. DNA repair synthesis in normal monocytes is blocked by aphidicolin, an inhibitor of DNA polymerase-alpha with respect to dCTP. Aphidicolin is also directly toxic to normal monocytes, but has no effect on nondividing lymphocytes or fibroblasts. Compared to most other cell types, monocytes and macrophages have very low dCTP pools, but abundant deoxycytidine kinase activity. This suggests that dCTP derived from salvage pathways is important for DNA repair in these cells. Consistent with this notion, exogenous deoxycytidine could partially protect monocytes from aphidicolin killing. The unexpected toxicity of aphidicolin toward normal human monocytes may be attributable to their high rate of spontaneous DNA strand break formation, to the importance of DNA polymerase-alpha for DNA repair in these cells, and to their minute dCTP pools.     

Distinctive properties of mammalian DNA polymerases.

DNA polymerase-alpha and -beta can be distinguished from one another by the differential effects of N-ethylmaleimide, KCl, ara-CTP and temperature, as well as on the basis of sedimentation. The sensitivity of DNA polymerase-beta to elevated temperatures as compared to DNA polymerase-alpha provides a new means of distinguishing between these two enzymes even in crude extracts and a possible probe for determining their function. DNA polymerase-alpha and -beta share several properties in common, including the ability to readily incorporate dUTP in place of dTTP. The Km for dUTP varies from 10 to 30 micron with different preparations of DNA polymerase-alpha and -beta. Thus, in mammalian cells, dUMP could be incorporated into DNA, and if excised by an endonuclease, would lead to discontinuities. Initial analyses of fidelity in direct comparative studies indicate that beta-class DNA polymerases are highly accurate in base selection when copying poly[d(A-T)]. Less than one molecule of dGMP is incorporated for every 12 000-45 000 molecules of dAMP and dTMP polymerized. DNA polymerase-alpha is somewhat less accurate, making one mistake for every 4000-10 000 correct nucleotides incorporated. Since both polymerases lack an exonucleolytic activity, this accuracy must be the result of selectivity for the complementary nucleotide by the polymerase.     

Stockpiling of DNA polymerases during oogenesis and embryogenesis in the frog, Xenopus laevis

The amounts of the various forms of DNA polymerase (alpha 1, alpha 2, beta, and gamma) have been determined in oocytes, eggs, and embryos of the frog, Xenopus laevis. During oogenesis the relative proportions and absolute levels of all forms changed dramatically. In stage I (early) oocytes, DNA polymerase-gamma, the "mitochondrial" polymerase, was the predominant form. During oocyte growth, DNA polymerase-alpha 1 and -alpha 2 increased by more than 100-fold, DNA polymerase-beta by 15-fold, and DNA polymerase-gamma by only 8-fold. During oocyte maturation and ovulation, the levels of all forms of DNA polymerase roughly doubled. The mature stage VI oocyte contained 5 orders of magnitude more DNA polymerase activity than is found in an individual somatic cell. DNA polymerase-alpha 1 and -alpha 2, the "replicative" polymerases, were the predominant forms in mature oocytes and ovulated unfertilized eggs. During fertilization, the relative proportions and absolute levels of the four forms remained constant. During subsequent stages of embryogenesis, the total amounts of DNA polymerase-alpha 1 and -alpha 2 declined slightly from cleavage through gastrulation, the stages of most rapid chromosomal DNA replication. The rapid increase in cell number during early embryogenesis establishes the same levels of DNA polymerase/cell as are present in adult somatic cells. After neurulation, the absolute levels of DNA polymerase-alpha 1 and -alpha 2 increased in proportion to increases in cell number. The absolute levels of DNA polymerase-beta remained constant, and the levels of DNA polymerase-gamma increased 2-fold throughout embryogenesis.     

Nuclear location of mammalian DNA polymerase activities.

Nuclei were isolated from monolayer cultures of mouse and human cells using a nonaqueous procedure of cell fractionation in which lyophilized cells were homogenized and centrifuged in 100% glycerol. In previous work we have shown that the nuclear pellet and cytoplasmic supernatant fraction contained 10% or less of the nucleic acids characteristic of the other cell fraction. Aqueous extracts made from fresh cultures and from nonaqueous material at each step of the fractionation procedure were assayed fro DNA polymerase activity. Activities were normalized to DNA contents of extracted material. Specific activity was preserved quantitatively through freezing and drying the cells, but was found to be unstable in glycerol suspensions with approximate half-lives and 1 h at 23 degrees and 4 h at 0-4 degrees. Activities were relatively stable at -25 degrees, however, so that by homogenizing only 15 min at 4 degrees and centrifuging at -25 degrees we preserved approximately 85% of the specific activity of fresh cultures in the nonaqueous nuclear fraction. Sedimentation analyses showed that the nuclear fraction contained both DNA polymerase-alpha and-beta in approximately the proportions expected if all polymerase activities were confined to the nucleus in living cells. DNA polymerase-alpha was found to be more unstable in glycerol suspensions than DNA polymerase-beta. Nuclear location of both activities was found in exponential cultures and in 3T3 mouse cultures synchronized in the G1 and S phases of the cell division cycle. We found no evidence for cytoplasmic factors affecting nuclear polymerase activities. We have concluded that the two major DNA polymerases are nuclear although one, DNA polymerase-alpha, frequently is present as a weakly bound nuclear protein.     

Purification and immunological characterization of DNA polymerase-alpha from human acute lymphoblastic leukemia cells

DNA polymerase-alpha was purified from the cytosol of blast cells of a patient with acute lymphoblastic leukemia by ammonium sulfate fractionation and successive column chromatographies. The purified enzyme had a specific activity of 2943 units/mg protein with activated calf thymus DNA as a template. The enzyme sediments under high-salt conditions as a homogeneous band at 7.2 S and free from other DNA polymerases (beta, gamma) and terminal deoxynucleotidyl transferase activity. The native molecular weight of the enzyme from gel filtration and glycerol gradient centrifugation was found to be 175 000. The values of Stokes radius (53 A), diffusion coefficient (4.05 x 10(-7) cm2/s) and frictional ratio (1.42) determined by gel filtration suggest that the native enzyme is compact and globular. Antibodies to DNA polymerase-alpha were raised in rabbits. These antibodies, partially purified by 50% ammonium sulfate saturation and Sephadex G-200 chromatography, gave one precipitin band on immunodiffusion and inactivate DNA polymerase-alpha-. This antibody preparation also inhibited, in vitro, the activity of DNA polymerase-alpha from calf thymus, phytohemagglutinin-stimulated normal human lymphocytes, as well as that from other leukemic cells. Thus, DNA polymerase-alpha from calf thymus and human leukemic cells resemble each other in antibody specificity.     

DNA polymerase, thymidine kinase and DNA synthesis in erythropoietic mouse spleen cells separated on bovine serum albumin gradients.

A single dose of erythropoietin stimulates DNA synthesis in the spleen of the polycythemic mouse with the maximum effect occurring 48 h after the hormone is administered. The increase in DNA synthesis is accompanied by morphologic evidence of increased erythropoiesis and by increases in the activities per cell of both thymidine kinase and cytoplasmic high molecular weight DNA polymerase-alpha. The activity of low molecular weight DNA polymerase-beta does not change significantly. Spleen cells from mice which had received either erythropoietin or saline 48 h previously were separated into 7 density classes on discontinuous bovine serum albumin gradients. Following the administration of erythropoietin, thymidine incorporation and thymidine kinase activity showed the greatest relative increases per nucleated cell in layers 3, 4 and 5 of the gradient. DNA polymerase-alpha showed the greatest increase in cells of the denser layers 5, 6 and 7. Each layer contained normoblasts and lymphocytes. The less well differentiated erythroid elements constituted a larger proportion of cells in layers of lower density. Increases in the rates of thymidine incorporation were better correlated with increases in thymidine kinase activity than with increases in DNA polymerase activities. Measurement of iron incorporation into heme confirm the morphological impression that the cell type responsible for increased thymidine incorporation and increased DNA polymerase-alpha activity is the young normblast.