Total DNA Polymerase Activity Research Articles

Microsatellite Instability in Arabidopsis Increases with Plant Development

Plant development consists of the initial phase of intensive cell division followed by continuous genome endoreduplication, cell growth, and elongation. The maintenance of genome stability under these conditions is the main task performed by DNA repair and genome surveillance mechanisms. Our previous work showed that the rate of homologous recombination repair in older plants decreases. We hypothesized that this age-dependent decrease in the recombination rate is paralleled with other changes in DNA repair capacity. Here, we analyzed microsatellite stability using transgenic Arabidopsis (Arabidopsis thaliana) plants that carry the nonfunctional β-glucuronidase gene disrupted by microsatellite repeats. We found that microsatellite instability increased dramatically with plant age. We analyzed the contribution of various mechanisms to microsatellite instability, including replication errors and mistakes of DNA repair mechanisms such as mismatch repair, excision repair, and strand break repair. Analysis of total DNA polymerase activity using partially purified protein extracts showed an age-dependent decrease in activity and an increase in fidelity. Analysis of the steady-state RNA level of DNA replicative polymerases α, δ, Pol I-like A, and Pol I-like B and the expression of mutS homolog 2 (Msh2) and Msh6 showed an age-dependent decrease. An in vitro repair assay showed lower efficiency of nonhomologous end joining in older plants, paralleled by an increase in Ku70 gene expression. Thus, we assume that the more frequent involvement of nonhomologous end joining in strand break repair and the less efficient end-joining repair together with lower levels of mismatch repair activities may be the main contributors to the observed age-dependent increase in microsatellite instability.

The effects of dehydroaltenusin, a novel mammalian DNA polymerase alpha inhibitor, on cell proliferation and cell cycle progression.

As described previously, a natural product isolated from fungus (Acremonium sp.), dehydroaltenusin, is an inhibitor of mammalian DNA polymerase alpha in vitro [Y. Mizushina, S. Kamisuki, T. Mizuno, M. Takemura, H. Asahara, S. Linn, T. Yamaguchi, A. Matsukage, F. Hanaoka, S. Yoshida, M. Saneyoshi, F. Sugawara, K. Sakaguchi, Dehydroaltenusin, a mammalian DNA polymerase alpha inhibitor, J. Biol. Chem. 275 (2000) 33957_33961]. In this study, we investigated the interaction of dehydroaltenusin with lipid bilayers using an in vitro liposome system, which is a model of the cell membrane, and found that approximately 4% of dehydroaltenusin was incorporated into liposomes. We also investigated the influence of dehydroaltenusin on cultured cancer cells. Dehydroaltenusin inhibited the growth of HeLa cells with an LD50 value of 38 microM, and as expected, S phase accumulation in the cell cycle. The total DNA polymerase activity of the extract of incubated cells with dehydroaltenusin was 23% lower than that of nontreated cells. Dehydroaltenusin increased cyclin E and cyclin A levels. In the analysis of the cell cycle using G1/S synchronized cells by employing hydroxyurea, the compound delayed both entry into the S phase and S phase progression. In a similar analysis using G2/M synchronized cells by employing nocodazole, the compound accumulated the cells at G1/S and inhibited entry into the S phase. Thus, the pharmacological abrogation of cell proliferation by dehydroaltenusin may prove to be an effective chemotherapeutic agent against tumors.

Long-term effects of caloric restriction initiated at different ages on DNA polymerases in rat brain

Caloric restriction (CR), has been shown to extend life span and delay both aging and the onset of a number of life-shortening diseases in laboratory animals. Defective DNA repair and DNA damage have been implicated as causal factors in many of these disease processes. CR appears to increase genetic stability by enhancing the DNA repair capacity and reducing the age-related accumulation of certain types of DNA damage. In the present study, we examined the effects of CR, imposed during four discrete periods of the life span of male Wistar rats, on the activity of total DNA polymerases of an aged brain which in turn may have a bearing on the process of aging. Our data indicates that CR had a greater stimulatory response on the activity of total DNA polymerases measured at 28 months of age in rats restricted from 6 to 28 months of age than in those restricted from postnatal day (PND) 7–21 which in turn was higher than those restricted from birth to PND 21. CR had differential effects in different regions of the brain. The enzyme activity of total DNA polymerases was significantly increased in hypothalamus and hippocampus. Cerebral cortex and corpus striatum are the regions which show no significant changes by CR regardless of age or duration of treatment while cerebellum registered a decrease during all periods of restriction. Modification of caloric intake alters total DNA polymerase activity and the extent of modification is both age and tissue dependent. Also, it appears that the effects of CR depend on when the paradigm is initiated, and are not solely a linear function of its duration.

Maize DNA polymerase 2 (an α-type enzyme) suffers major damage after seed deterioration

AbstractDNA polymerase 2 holoenzyme is an α-type polymerase present as a major activity in 24-h germinated maize embryo axes. At this time, several of the polypeptides that compose this multisubunit enzyme are phosphorylated. During the germination of artificially deteriorated maize embryo axes, total DNA polymerase activity decreases by around 50% when compared with activity in non-deteriorated axes. DNA polymerase 2 appears to be damaged by the deterioration treatment, greatly reducing its activity. This decrease in activity is related to protein degradation during germination since the amount of both the holoenzyme and the different polymerase 2 subunits is reduced as imbibition progresses. The decrease in amount is more dramatic for some of the subunits than for others, i.e., the amount of the 90-kDa catalytic subunit is reduced by about half, whereas all other subunits totally disappear at different times during germination. The phosphorylation state of the different polypeptides is also greatly affected.

Regulation of DNA repair and replication in proliferating FL cells

In this paper, we show that the rate of total DNA synthesis increases twice during the Friend erythroleukemia (FL) cell culture growth cycle. The first increase takes place at the threshold of the logarithmic (early) phase of growth, while the second peak takes place in the mid-logarithmic (late) phase. This bi-phasic pattern can be reproduced in both normal and dimethylsulfoxide (DMSO)-differentiating cells, using either [3H]-thymidine or [14Cl-methyl-L-methionine as precursors for DNA labelling. In normal cells, the non-replicating strands incorporate some radioactivity originating in [3H]-thymidine, essentially in the early phase, while their labelling is insignificant in the late phase. In UV-irradiated cells, the mechanism yielding repair patches (RPs) contained by the same non-replicating strands is highly facilitated in the earlier stage in comparison with its functioning at the later stage. In contrast, the [3H]-thymidine labelling of the semiconservatively newly replicating strands is insignificant at the early stage and more substantial later, as expected. In UV-irradiated cells, the slightly decreased [3H]-thymidine labelling of replicating strands is probably explained by the concomitant small percentage of cell death. The chronology of the optimal rates of early repair and later replication is quite well confirmed by a bi-phasic regulation of the total DNA poly-merase (pol) activity: while a peak of N-ethylmaleimide (MalNEt)-resistant pols activity (accounting for base excision-repair) is seen early on, a peak of MalNEt-sensitive polα activity (accounting for replication) is found later. DMSO strikingly inhibits both the rate of the early base excision-repair (either exploiting [3H]-thymidine or [14C]-methyl-L-methionine) and the early MalNEt-resistant pols activity. It does not, however, inhibit the late replication and the late MalNEt-sensitive polα activity, when hemoglobin (Hb) synthesis is heavily induced. The time-shift of late replication in DMSO-differentiating cells is probably explained by a delay of theS-phase, as also suggested by the net decrease in the amount of DNA per nucleus during the second half of the culture growth cycle.

Analysis of DNA polymerase activity in Petunia protoplasts treated with clastogenic agents

Clastogenic agents, i.e. agents that can induce chromosome or DNA breakage, have been shown to enhance the rale of direct gene transfer to protoplasts. The effect was analysed at the enzymatic level using protoplast homogenates as well as intact protoplasts. For that purpose existing procedures were modified to enable measurement of DNA polymerase in vivo. In the system used, external DNA was able to enter the cells without the addition of membrane‐permeabilizing compounds. When comparing total DNA polymerase activity of protoplasts irradiated with X‐rays or UV‐light with that of untreated cells we did not observe significant differences. Incubation of protoplasts with high doses of bleomycin affected total DNA polymerase activity negatively. but dideoxythymidine triphosphate‐sensitive activity was not influenced. We conclude that the DNA strand‐breaks induced by low doses of X‐rays. UV‐light or bleomycin do not increase the total or the repair‐DNA polymerase activity and. therefore. that the increase in the transformation rates after DNA strand‐breaking is not preceded by enhanced DNA polymerase activity.

Changes in the activities of DNA polymerases in growing rat lungs.

We hypothesized that cellular proliferation and the capacity to repair DNA damage in the lung might differ during the pre- and postnatal periods, because the lung is exposed to higher oxygen concentrations and/or various mutagens after birth. In order to test this hypothesis, changes in DNA content and the activities of DNA polymerase alpha and beta were studied in the lungs of 1-day prenatal to 42-day postnatal rats. Total DNA polymerase activity reached its highest level at 1 day prenatal and 1 day after birth. The activity decreased exponentially by 28% up to 14 days of age, a change inversely related to the change in DNA content. The change in total DNA polymerase activity agreed closely with the change in DNA polymerase alpha activity, but not the activity of the beta form, although small elevations in both DNA polymerase alpha and beta were observed on day 3, possibly reflecting the mechanical effect of delivery. The activity of DNA polymerase beta remained relatively constant from 1 day before birth to 21 days after birth, varying by only about 5%. From these results, it is concluded that: (1) cellular proliferation in the lung is most active during the first 2 weeks after birth as supported by the increases in DNA polymerase alpha activity and DNA content, and (2) anticipating the oxygen enriched atmosphere after birth, the level of DNA polymerase beta, involved in the DNA repair system, is already elevated during the prenatal period and remains constant throughout the postnatal period.

Induction of DNA polymerase activities in the regenerating rat liver

The levels of DNA polymerase alpha, DNA polymerase delta, and its accessory protein, proliferating cell nuclear antigen (PCNA) were examined in the regenerating rat liver. The levels of DNA polymerase alpha and delta activities in regenerating liver extracts were determined by the use of the DNA polymerase alpha specific inhibitor, BuAdATP [2-(p-n-butylanilino)-9-(2-deoxy-beta-D-ribofuranosyl) adenine 5'-triphosphate], and monoclonal antibodies. These reagents showed that the total DNA polymerase activities increased ca. 4-fold during regeneration and that the fraction of DNA polymerase delta activity at the peak was 40% of the total DNA polymerase activity. Immunoblots and inhibition studies using specific antibodies showed that DNA polymerase delta and epsilon and PCNA were concomitantly induced after partial hepatectomy. The levels of both DNA polymerase delta and epsilon and PCNA reached their maxima at 24-36 h post hepatectomy, i.e., at the same time that in vivo DNA synthesis reached its peak. Partial purification and characterization of DNA polymerases delta and epsilon from the regenerating rat liver were also performed. These observations suggest that the variation of DNA polymerase delta and epsilon and PCNA during liver regeneration is closely related to DNA synthesis and is consistent with their involvement in DNA replication.

Aphidicolin hypersensitive mutant of chinese hamster V79 fibroblasts that underproduces DNA polymerase-? antigen

Aphidicolin is a specific inhibitor of DNA polymerase-alpha and -delta from eukaryotic cells. Because of the specificity of this inhibitor, it is potentially a useful probe for the detailed studies of the function of these polymerases. DNA polymerase-alpha mutants isolated on the basis of resistance to aphidicolin have been described. We have isolated four variants that exhibit hypersensitivities to aphidicolin (Aphhs) from Chinese hamster V79/743X fibroblasts. These variants are designated aphhs-1, aphhs-2, aphhs-3 and aphhs-4. We reported here results of studies involving immunochemical characterization. The Aphhs phenotype in all mutants was stable for at least 30 days in the absence of selection pressure. The dCTP pools in the 743X and Aphhs cell lines were not significantly different. The level of total DNA polymerase activity in the crude extract from aphhs-2 cells was 30% of that observed in the parental 743X clone. We developed a method to quantitate DNA polymerase-alpha antigen at single cells in situ using monoclonal antibody SJK 132-20 and fluorescence pseudocolor image. We found that the antigen of DNA polymerase-alpha in aphhs-2 was 30-50% of that in the parental 743X cells. The underproduction of the antigen of DNA polymerase-alpha provides a basis for the observed Aphhs phenotype. Possible mechanisms for the underproduction of DNA polymerase-alpha in aphhs-2 clone are presented.

Cell cycle dependent activities of DNA polymerases alpha and delta in Chinese hamster ovary cells.

The activities of DNA polymerases alpha and delta, in extracts from Chinese hamster ovary (CHO) cells, were assayed in order to determine whether these polymerases are regulated during the cell cycle. An exponential population of CHO cells was separated into enriched populations of G-1, S, and G-2/M phases of cell cycle by centrifugal elutriation. Total cell homogenates from each population were assayed for DNA polymerase activity by measuring labeled nucleotide incorporation into the exogenous templates oligo(dT).poly(dA) and DNase I activated calf thymus DNA. In these experiments, specific DNA polymerase inhibitors were added to assays of the cellular extracts to allow for the independent measurement of activities of DNA polymerases alpha and delta. Comparisons of total DNA polymerase activity from cellular extracts, sampled from each portion of the cell cycle, demonstrated no significant change with respect to the concentration of total protein. However, results indicate that the activity of DNA polymerase delta increases with respect to that of DNA polymerase alpha in the G-2/M portion of the cell cycle. This difference in relative activities of DNA polymerases alpha and delta suggests a coordinate regulation of a specific species of DNA polymerase during the cell cycle.

Changes in DNA polymerases .alpha., .beta., and .gamma. during the replicative life span of cultured human fibroblasts

DNA polymerases from IMR-90 human diploid fibroblasts at various passage levels and from HeLa cells were purified and fractionated into alpha 1, alpha 2, alpha 3, beta, and gamma species and subspecies, and than the accuracy with which each one copied synthetic template-primers was measured in the presence of Mn2+ or Mg2+. All activities from fibroblasts of later population doubling levels incorporated noncomplementary triphosphates more frequently than did the same polymerase type from earlier population doubling levels. HeLa polymerase activities copied several different templates in the presence of Mn2+ with greater fidelity than enzymes from fibroblasts of population doubling level 27 or greater. The total DNA polymerase activity extracted from IMR-90 cells decreased with increasing population doubling levels. The alpha-polymerase activity generally declined with increasing population doubling levels, while beta-polymerase activity remained relatively constant, except at the very end of the cellular replicative life span. In addition, the amounts of alpha 2 and alpha 3 became progressively lower relative to alpha 1, and a new alpha-type polymerase activity, alpha 0, appeared upon diethylaminoethylcellulose chromatography. HeLa cells also contained three alpha species, though two of them eluted from diethylaminoethylcellulose at higher phosphate concentrations than alpha species from fibroblasts. Postconfluent IMR-90 cells of population doubling level 21 had a decreased level of alpha-polymerase relative to that recovered from rapidly growing cells. This polymerase activity had some chromatographic properties similar to enzyme from late-passage cells. In addition, the alpha-, beta-, and gamma-polymerases from these cells had decreased fidelities relative to those isolated from subconfluent cells.

DNA polymerase alpha from the nuclear matrix of cells infected with simian virus 40.

The nuclear matrix prepared from normal, simian virus 40 (SV40)-infected, and SV40-transformed cells contained DNA polymerase activities. Approximately 12% of the total DNA polymerase activities in isolated nuclei remained with the nuclear matrix. alpha-polymerase was the major matrix DNA polymerase activity as judged by sensitivity to various inhibitors: aphidicolin, dideoxy-TTP, and N-ethylmaleimide. Approximately 2-4 fold higher DNA polymerase activity was detected in matrices obtained from lytically infected and virus-transformed cells than that found in normal cells. In lytically infected cells, 30-50% of the matrix-bound DNA polymerase activity solubilized by sonication co-sedimented with majority of the matrix T-antigen, and was co-precipitated with anti-T sera. The results suggest that alpha-polymerase and viral T-antigen may form a functional complex in the matrix.

DNA synthesis catalysed by endogenous templates and DNA-dependent DNA polymerases in spermatogenic cells from rat

The activity levels of DNA polymerases α and β have been measured by autoradiography in squash preparations from rat testis of sexually mature animals. Similar results were obtained with ‘fixed’ samples (dipped in acetone: ethanol for 5 min at 25 °C) or ‘unfixed’ samples (frozen in liquid nitrogen and freeze-dried). The activities of DNA polymerases α and β in situ were distinguished by differential assay conditions and by selective inhibition with compounds such as N-ethylmaleimide and aphidicolin. Using the endogenous chromatin as template, maximal activity for both enzymes was obtained in the presence of all four deoxyribonucleoside triphosphates, MgCl 2 and ethylene glycol. When DNA polymerase activities in several predominant testicular cell types (pre-leptotene primary spermatocytes, pachytene primary spermatocytes, round spermatids and elongated spermatids) were quantitatively compared, on a per cell basis, the following percentage distribution was observed: Pre-leptotene primary spermatocyte % Pachytene primary spermatocyte % Round spermatid % Elongated spermatid % DNA polymerase α 25 42 30 3 DNA polymerase β 29 34 36 1 Although elongating spermatids generally contained a small proportion of the total DNA polymerase activities assayed (1–3%), occasionally elevated levels of activity of both DNA polymerases α and β were observed at stage 11 coinciding with the onset of the histone transition, a temporal event leading to marked change in template composition. Significant levels of DNA polymerase β were also detected in the proximal region of the elongated spermatids at stage 19 of spermiogenesis.

Androgen-induced replication of prostate chromatin DNA: Isolation and electron microscopic visualization of replicative in intermediates

A procedure originally developed to isolate transcriptionally active from less active rat ventral prostate chromatin, employing minimal shear and centrifugation through a dense sucrose gradient, was used to separate prostate chromatin actively synthesizing DNA from total chromatin. It was verified that maximum DNA synthesis in ventral prostates of rats 6 days after castration occurred after administration of testosterone propionate daily for 3 days. When minced ventral prostates from such animals were incubated with [ 3H]thymidine, and the ‘heavy’ and ‘light’ chromatin fractions were separated by sucrose gradient centrifugation, most radioactive DNA was present in the ‘light’ fraction at the top of the gradient. Incorporation was due to DNA synthesis and not to repair, as judged by inhibition with N-ethylmaleimide. Results of in vitro and in vivo thymidine pulse-chase experiments were consistent with initial labelling of DNA-in a replication complex and subsequent sequestration of radioactive DNA in forms resistant to release by the preparative procedure. Although about half the estimtated total endogenous DNA polymerase activity detected in vitro was present in the heavy fraction, the apparent specific activity of the enzyme in the ‘light’ fraction was 5-times as high. Lastly, when equal concentrations of DNA from the separated chromatin fractions were shadowed with platinum-palladium and examined by electron microscopy, 5-times as many Y-shaped structures were seen in the light fraction. This procedure facilitates the isolation of enzymatically active DNA structures undergoing semiconservative replication and study of their subsequent molecular ‘processing’ into forms no longer susceptible to separation by this comparatively gentle method of chromatin preparation. Since the method also yields transcriptionally active chromatin fractions from rat liver and Chinese hamster ovary cell nuclei, it should be applicable to the study of DNA synthesis in these and many other cells.

Enrichment of DNA polymerase III activity in a DNA membrane complex purified from Pneumococcus: the possible existence of subcomplexes.

Three DNA polymerase activities, one related to DNA pol III, have been extracted from a DNA membrane complex purified from Streptococcus pneumoniae. DNA pol III was purified 3300-fold, DNA pol II 2800-fold and DNA pol I 1800-fold. Based on inhibition analysis with a drug known to inhibit DNA pol III activity in Gram positive organisms. 6(p-hydroxyphenyl azo) uracil (HpU), 55% of the total DNA polymerase activity is represented by pol III. In contrast, only 3-5% of the total DNA polymerase activity is inhibited by HpU in crude extracts. The purification of the DNA membrane complex from pneumococcus is modified from an earlier procedure (Firshein 1972). The modified procedure results in the separation of three distinct DNA-protein-phospholipid subcomplexes of which the one described above contains most of the radioactivity derived from cells pulsed for a short time with (3H)-thymidine. Proteins are involved in binding DNA in each complex and the conformation of DNA in each complex may be different. All of the subcomplexes contain DNA polymerase activity partially sensitive to HpU. These results provide direct evidence for the structural integrity of a complex that may be involved in DNA replication in vivo.

DNA-dependent DNA polymerase species in male germ cells of the mouse

Quasi-homogeneous fractions of male mouse germ cells at definite stages of meiosis and spermiogenesis were obtained by using a separation method based on sedimentation velocity in an albumin gradient. In the various cell types, the total DNA-dependent DNA polymerase activity was determined, and the major enzymatic forms were characterized. The DNA polymerase species present in premeiotic, meiotic and post-meiotic cells were analyzed by glycerol gradient sedimentation. Two types of DNA polymerase were identified in fractions enriched in spermatogonia and preleptotene spermatocytes. One showed a sedimentation coefficient of about 7.5 S and was sensitive to N-ethylmaleimide (NEM); the other exhibited a sedimentation coefficient between 3 and 4 S and was resistant to NEM. On the basis of their sedimentation coefficients, their sensitivity to NEM and their template specificities, these 2 enzymes were identified respectively as α and β DNA polymerases as reported in mammals. The gradient analysis performed on fractions enriched in meiotic and post-meiotic cells revealed the presence of DNA polymerase β only. A quantitative analysis showed that the activity of the DNA polymerase β reaches a maximum at middle-late pachytene stage and then drops gradually during spermiogenesis. Although any conclusion as to the biological role of this high level of DNA polymerase activity in pachytene spermatocytes is premature, it is tempting to suggest that this enzyme is involved in meiotic recombination.

DNA polymerase alpha and beta in the California urchin.

DNA polymerase alpha and beta were identified in the urchin, Strongylocentrotus purpuratus. The DNA polymerase beta sedimented at 3.4 S, constituted 5% of total DNA polymerase activity, and was resistant to N-ethylmaleimide and high ionic strength. The polymerase alpha sedimented at 6--8 S, was inhibited by N-ethylmalemide or 0.1 M (NH4)2SO4, and was dependent upon glycerol for preservation of activity. Both the polymerases alpha and beta were nuclear associated in embryos. The DNA polymerase alpha was markedly heterogeneous on DEAE-Sephadex ion exchange and showed three modal polymerase species. These polymerase alpha species were indistinguishable by template activity assays but the DNA polymerase associated ribonucleotidyl transferase (Biochemistry 75 : 3106-3113, 1976) was found predominantly with only one of the DNA polymerase alpha species.

Cellular localization of DNA polymerase activities in full-grown oocytes and embryos of Xenopus laevis

In full-grown oocytes of Xenopus laevis more than 80 % of the total DNA polymerase activity is found in the germinal vesicle (nucleus) and only about 8% in the cytoplasm. The intracellular distribution of the multiple DNA polymerase forms has been studied in oocytes and in embryonic cells. The oocyte nucleus contains a major DNA polymerase species, sedimenting at about 7S, and a minor species sedimenting at about 5S. These enzymes are comparable, respectively, with the DNA polymerases α and β described in other biological systems. In the oocyte cytoplasm, besides a small amount of the 7S form, an 8–9S DNA polymerase activity is also detectable. In the nuclei of embryonic cells, in addition to the DNA polymerase forms present in the oocyte nucleus, a new major form which seems specific for the eggs and embryos is detectable by DEAE chromatography.

Characterization of DNA polymerase associated with nuclear membrane fractions from uninfected and adenovirus 2-infected KB cells

We have isolated a nuclear membrane fraction from KB cells infected with human adenovirus 2 that synthesizes exclusively small viral DNA chains (approx. 9 S) in vitro (Yamashita, T., Arens, M. and Green, M. (1975) J. Biol. Chem. 250, 3273–3279). The DNA polymerase activity present in the adenovirus 2 DNA-nuclear membrane complex was purified through chromatography on phosphocellulose and DEAE-cellulose, glycerol gradient centrifugation and DNA-cellulose chromatography. A single peak of enzymatic activity sedimented in glycerol gradients at about 6.7 S which corresponds to a molecular weight of 125 000. The enzyme preparation in the step of glycerol gradient centrifugation utilized activated calf thymus, KB cell and adenovirus 2 DNA as template-primer in the presence of Mg 2+; K m values for these DNAs were 5.5, 4.0, and 0.8 μg/ml, respectively. The partially purified enzyme preparation was characterized by several criteria which were compared to the properties of the three major mammalian DNA polymerases, α, β, and γ. On the basis of template-primer preference, effect of salt, inhibition by N- ethylmaleimide and K m for dTTP, the DNA polymerase activity from the membrane complex can be distinguished from the α and β DNA polymerases. The elution profile from DNA cellulose revealed a minor peak (I) and a major peak (II) of DNA polymerase activity utilizing poly(A) · (dT) 10 as template-primer in the presence of Mn 2+. Peak II from DNA cellulose, which contained about 90% of the total DNA polymerase activity eluted from the column, was 2–3 times as active with poly(A) · (dT) 10 as template-primer in the presence of Mn 2+ than with activated calf thymus DNA in the presence of Mg 2+. On the other hand, peak I had a low ratio of poly(A) · (dT) 10 to activated calf thymus DNA activity. DNA polymerase was also purified from the nuclear membrane fraction of uninfected KB cells by the same procedures as those used in enzyme purification from the adenovirus 2 DNA-nuclear membrane complex. A minor peak and a major peak of DNA polymerase activity utilizing poly(A) · (dT) 10 as template primer in the presence of Mn 2+ were again observed that eluted from DNA cellulose at the same KCl concentrations as peak I and II from adenovirus 2-infected cells. The enzymes of the nuclear membrane fraction of uninfected KB cells could not be differentiated from the enzymes of the adenovirus 2 DNA-nuclear membrane complex through any of the purification steps nor by their template specificities. These results indicate that the predominant enzyme in the adenovirus 2 DNA-nuclear membrane complex and in the KB cell nuclear membrane complex belongs to the class of DNA polymerase γ.

DNA-metabolizing Enzymes in Normal Human Lymphoid Cells. VI. Induction of DNA Polymerases α, β, and γ Following Stimulation With Phytohemagglutinin

At least three distinct DNA polymerases, named alpha, beta, and gamma, have been isolated from normal mammalian cells. The function of these enzymes in regard to DNA replication and repair remains unclear. Stimulation of blood lymphocytes with the plant mitogen phytohemagglutinin (PHA), is known to increase total DNA polymerase activity. In this study, we measured the change of each of these activities as lymphocytes intered a mitotic cycle. Aliquots of a pool of normal human blood lymphocytes were incubated with PHA for 0, 24, 48, and 72 hr, respectively, and the various DNA polymerase activities quantitated at each point. No significant DNA polymerase activity was detected in unstimulated cells. Low levels of polymerase beta were found at 24 hr. The average DNA content per cell doubled between 24 and 48 hr, and during this interval all three DNA polymerases increased to easily detectable levels. By far the greatest fractional increase in activity of all three polymerases was seen between 48 and 72 hr, after the average doubling of cellular DNA. In summary, these blood lymphocytes lack significant levels of DNA polymerases; stimulation with PHA induces all three of the major DNA polymerase species. In both these respects, these cells differ from other proliferating mammalian cell systems. The possible significance of this difference is discussed.