Control of DNA polymerase α, β and γ activities in heat- and cold-sensitive mammalian cell-cycle mutants

Two heat-sensitive (arrested in G₁ at 39.5°C) and two cold-sensitive (arrested in G₁ at 33°C) clonal cell-cycle mutants of the murine P-815-X2 mastocytoma line were tested for DNA polymerase α, β and γ activities. After transfer of mutant cells to the respective nonpermissive temperature, DNA polymerase α activities decreased more slowly than relative numbers of cells in S phase. Furthermore, numbers of DNA-synthesizing cells decreased to near-zero levels, whereas polymerase α activities in arrested cells were as high as 15–40% of control values. After return of arrested cells to the permissive temperature, polymerase α activities increased essentially in parallel with relative numbers of cells in S phase. In contrast to the changes in thymidine kinase (Schneider, E., Müller, B. and Schindler, R. (1983) Biochim. Biophys. Acta 741, 77–85), the decrease of polymerase α during entry of cells into proliferative quiescence thus appears to be under rather relaxed control, while after return of arrested cells to the permissive temperature the increase in polymerase α is tightly coupled with reentry of cells into S phase. For DNA polymerase β and γ activities, no obvious correlation with changes in the proliferative state of cells was detected.     

Control of ribonucleotide reductase in heat- and cold-sensitive mammalian cell-cycle mutants

Two heat-sensitive (reversibly arrested in G1 phase at 39.5 degrees C, multiplying at 33 degrees C) and two cold-sensitive (reversibly arrested in G1 phase at 33 degrees C, multiplying at 39.5 degrees C) cell-cycle mutants of the P-815-X2 murine mastocytoma line were tested for ribonucleotide reductase activity, using cells made permeable to nucleotides. After transfer of the heat-sensitive mutant cells to 39.5 degrees C, ribonucleotide reductase activity, similar to thymidine kinase (Schneider, E., Müller, B. and Schindler, R. (1983) Biochim. Biophys. Acta 741, 77-85), but unlike DNA polymerase alpha (Schneider, E., Müller, B. and Schindler, R. (1985) Biochim. Biophys. Acta 825, 375-383), decreased rapidly and in parallel with numbers of cells in S phase, whereas in the cold-sensitive mutant cells brought to 33 degrees C, ribonucleotide reductase activity decreased approx. 8 h later than numbers of DNA-synthesizing cells. When arrested heat- or cold-sensitive mutant cells were returned to the permissive temperature, ribonucleotide reductase activities, similar to DNA polymerase alpha and to thymidine kinase in heat-sensitive mutants, increased essentially in parallel with reentry of cells into S phase, whereas the increase in thymidine kinase activity in the cold-sensitive mutants was previously shown to occur approx. one cell-cycle time later. This indicates that ribonucleotide reductase and thymidine kinase are coordinately expressed in the heat-sensitive, but independently regulated in the cold-sensitive mutants.     

A temperature-sensitive mutation affecting S-phase progression can lead to accumulation of cells with a G2 DNA content

Cultures of ts BN75, a temperature-sensitive mutant of BHK 21 cells, show a gradual biphasic drop in [3H]thymidine incorporation together with an accumulation of cells having a G2 DNA content when incubated at 39.5 degrees. However, when higher (41 degrees - 42 degrees) nonpermissive temperatures were used, the major block was in S-phase DNA synthesis. The cultures of ts BN75 shifted to 42 degrees at the start of the S phase, cell-cycle progress was arrested in the middle of S, while under these conditions wild-type BHK cells underwent at least one cycle of DNA synthesis. When ts BN75 cells growth-arrested at high temperature with a G2 DNA content were shifted to the permissive temperature (33.5 degrees C), the restart of DNA synthesis preceded the appearance of mitotic cells. These data suggest that the ts defect of ts BN75 cells might affect primarily the S phase of the cycle rather than the G2 phase.     

Isolation of temperature-sensitive cell cycle mutants from mouse FM3A cells. Characterization of mutants with special reference to DNA replication

A large number of mutants that are temperature sensitive (ts) for growth have been isolated from mouse mammary carcinoma FM3A cells by an improved selection method consisting of cell synchronization and short exposures to restrictive temperature. The improved method increased the efficiency of isolating DNA ts mutants, which showed a rapid decrease in DNA-synthesizing ability after temperature shift-up. Sixteen mutants isolated by this and other methods were selected for this study. Flow microfluorometric analysis of these mutants cultured at a nonpermissive temperature (39 degrees C) for 16 h indicated that five clones were arrested in the G1 to S phase of the cell cycle, six clones were in the S to G2 phase, and two clones were arrested in the G2 phase. The remaining three clones exhibited 8C DNA content after incubation at 39 degrees C for 28 h, indicating defects in mitosis or cytokinesis. These mutants were classified into 11 complementation groups. All the mutants except for those arrested in the G2 phase and those exhibiting defects in mitosis or cytokinesis showed a rapid decrease in DNA synthesis after temperature shift-up without a decrease in RNA and protein synthesis. The polyomavirus DNA cell-free replication system, which consists of polyomavirus large tumor antigen and mouse cell extracts, was used for further characterization of these DNA ts mutants. Among these ts mutants, only the tsFT20 strain, which contains heat-labile DNA polymerase alpha, was unable to support the polyomavirus DNA replication. Analysis by DNA fiber autoradiography revealed that DNA chain elongation rates of these DNA ts mutants were not changed and that the initiation of DNA replication at the origin of replicons was impaired in the mutant cells.     

Dominant versus recessive behavior of a cold- and a heat-sensitive mammalian cell cycle variant in heterokaryons

A heat-sensitive (hs, arrested at 39.5 degrees C, termed 21-Ta) and a cold-sensitive (cs, arrested at 33 degrees C, termed 21-Fb) clonal cell cycle variant were isolated from the same clone of the P-815 murine mastocytoma line. At the respective nonpermissive temperatures, both the hs and the cs variant were reversibly arrested in G1 phase, and numbers of cells forming colonies upon reincubation at the permissive temperature remained nearly constant for at least 6 days. Cells arrested in G1 by incubation at the respective nonpermissive temperatures were fused to cells of another P-815 clone (31-S) that had been arrested by serum deprivation. Upon reincubation in medium containing 10% serum for 48 h at 39.5 degrees C, 21-Ta x 31-S heterokaryons, similar to 31-S x 31-S homokaryons, entered the S phase, whereas at 33 degrees C, 21-Fb x 31-S heterokaryons, similar to 21-Fb x 21-Fb homokaryons, remained arrested in G1, indicating a recessive expression of the hs and a dominant expression of the cs phenotype.     

Stimulatory and inhibitory effects of extracts from mammalian cell-cycle mutants on DNA replication in partially lysed cells

Heat-sensitive (arrested at 39.5 degrees C, multiplying at 33 degrees C) and cold-sensitive (arrested at 33 degrees C, multiplying at 39.5 degrees C) cell-cycle mutants of the P-815-X2 murine mastocytoma line were used for the preparation of cell extracts. These were tested for their effects on DNA synthesis in 'gently lysed cells' (obtained by treatment with 0.01% Brij-58) or 'highly lysed cells' (obtained by treatment with 0.1% Brij-58). Gently lysed cells prepared from proliferating P-815-X2 or mutant cells incorporated [3H]dTTP efficiently, while highly lysed cells exhibited a low level of [3H]dTTP incorporation which was markedly increased by the addition of extracts from proliferating cells. Extracts prepared from arrested mutant cells, however, were found to inhibit DNA synthesis by gently and highly lysed cells prepared from proliferating cells. After return of arrested mutant cells to the permissive temperature, stimulating activity in cell extract reappeared at the time of reentry of cells into S phase. Both stimulatory and inhibitory activities were associated with material(s) of molecular weight above 25 000, but differed in heat sensitivity and in sensitivity to immobilized proteinase and ribonuclease. Extracts from arrested cells counteracted the stimulating effects of extracts from proliferating cells with kinetics suggesting competitive interaction between stimulating and inhibitory factors.     

Further characterization of a murine temperature-sensitive mutant, tsFT20 strain, containing heat-labile DNA polymerase alpha-activity

tsFT20 cells, which have temperature-sensitive DNA polymerase alpha-activity, were characterized mainly at the cellular level. The cells lost their ability to synthesize DNA immediately after a shift to non-permissive temperature. The extent of decrease in the activity of DNA polymerase alpha in whole-cell extracts was the same as that of the decrease in the DNA replication ability determined by [3H]thymidine incorporation. At 39 degrees C, tsFT20 cells lost most of their colony-forming ability in one doubling time (16 h). The cells could not grow at higher than 38 degrees C, but could grow at 37 degrees C. When tsFT20 cells were synchronized at the G1/S boundary and incubated at 39 degrees C, they could not complete the S phase, ceasing cell cycle progression in mid-S phase. A temperature shift (33 degrees C----39 degrees C) experiment indicated that the whole S phase was temperature-sensitive, whereas the G2 and M phases were not. These results confirmed that DNA polymerase alpha plays a key role in DNA replication in mammalian cells.     

Participation of mitochondrial proliferation in morphological differentiation of murine mastocytoma cells

Proliferation of a cold-sensitive cell-cycle mutant isolated from an undifferentiated murine mastocytoma line is reversibly arrested at the nonpermissive temperature of 33 degrees C, and the arrested cells undergo morphological differentiation as expressed by the formation of metachromatic granules. Following transfer of these mutant cells from the permissive temperature of 39.5 to 33 degrees C, a transient increase in both cytochrome c oxidase and DNA polymerase gamma was observed, the ratio of total mitochondrial volume to cell volume nearly doubled within 6 days, and numbers of mitochondrial cross-sections per cellular cross-section as determined in electron micrographs underwent a threefold increase. Addition of chloramphenicol (100 micrograms/ml) to the mutant cell cultures 6 days prior to transfer from 39.5 to 33 degrees C prevented the increase in the ratio of total mitochondrial to cell volume. Furthermore, chloramphenicol markedly inhibited the increase in granule number per cell that normally is observed after transfer of cultures to 33 degrees C or during treatment with 1 mM butyrate, suggesting that mitochondrial proliferation may be an obligatory step in the process of morphological differentiation of these mastocytoma cells.     

Study of dogfish (Scyliorhinus caniculus) deoxyribonucleic acid polymerase alpha and beta. Extraction, separation, characterization and changes during spermatogenesis.

DNA polymerase activity was extracted from testis cells of the dogfish Scyliorhinus caniculus. On a sucrose gradient, two main peaks could be separated, corresponding to DNA polymerases beta (3.8 S) and alpha (7.5 S). DNA polymerase gamma could also be detected when poly(A) . (dT)12 was used as template. The properties of alpha and beta polymerases of this primitive vertebrate were similar to those generally described, especially in mammals. The beta enzyme was highly sensitive to N-ethylmaleimide, however, and could use poly(dT) . poly(A) as template. Polymerase alpha was present in spermatogonia, spermatocytes and spermatids. Activity was maximal in spermatocytes. DNA polymerase beta was present in all testis cells with similar activities in spermatogonia and spermatocytes. Decreased activities were observed during spermiogenesis. Some activity remained associated with the chromatin fraction of mature sperm cells.     

Effects of temperature changes on thymidine kinase in heat- and cold-sensitive cell-cycle mutants and ‘wild-type’ murine P-815 cells

Two heat-sensitive (arrested in G1 at 39.5°C) and two cold-sensitive (arrested in G1 at 33°C) clonal cell-cycle mutants that had been isolated from the same clone (K 21), of the murine mastocytoma P-815 cell line, were tested for thymidine kinase (EC 2.7.1.21) activity. After shift of mutant cells to the nonpermissive temperature, thymidine kinase activity decreased, and minimal levels (i.e., less than 3% of those observed for ‘wild-type’ K 21 cells at the respective temperature) were attained within 16 h in heat-sensitive and after 3–4 days in cold-sensitive mutants, which is in good agreement with kinetics of accumulation of heat-sensitive and cold-sensitive cells in G1 phase. After return of arrested mutant cells to the permissive temperature, thymidine kinase of heat-sensitive cells increased rapidly and in parallel with entry of cells into the S phase. In cultures of cold-sensitive cells, however, initiation of DNA synthesis preceded the increase of thymidine kinase activity by approx. one cell-cycle time. Thymidine kinase activities in revertants of the heat-sensitive and cold-sensitive mutants were similar to those of ‘wild-type’ cells. In ‘wild-type’ K 21 cells incubated at 39.5°C, thymidine kinase activity was approx. 30% of that at 33°C. This difference is attributable, at least in part, to a higher rate of inactivation of the enzyme at 39.5°C, as determined in cultures incubated with cycloheximide. The rapid increase of thymidine kinase activity that occurred after shift of K 21 cells and of arrested heat-sensitive mutant cells from 39.5°C to 33°C was inhibited by actinomycin D and cycloheximide.     

Immunocytochemical localization of chick DNA polymerases alpha and beta +

An immunofluorescent method using specific antibodies was employed to detect DNA polymerases alpha and beta in chick cells. With monoclonal antibodies produced by four independent hybridoma clones, most of the DNA polymerase alpha was shown to be present in nuclei of cultured chick embryonic cells. With a polyclonal, but highly specific, antibody against DNA polymerase beta, this enzyme was also shown to be present in nuclei. DNA polymerase alpha was detected in proliferating cells before cell contact and in lesser amount in resting cells after cell contact, indicating that its content is closely correlated with cell proliferation. On the other hand, similar amounts of DNA polymerase beta were detected in proliferating and resting cells. Furthermore, DNA polymerase beta was detected in nuclei of most cells, while DNA polymerase alpha was detected only in large round nuclei in seminiferous tubules of chick testis. DNA polymerase alpha is presumably present in cells that are capable of DNA replication, and during the cell cycle it seems to remain in the nuclei during the G1, S, and G2 phases, but to leave from condensed chromatin for the cytoplasm during the mitotic phase.     

Characteristic alteration in the nuclear DNA polymerase activity during the cell division cycle of Saccharomyces cerevisiae

Specific activity of the intranuclear DNA polymerase in cdc-mutant cells of Saccharomyces cerevisiae was found to be characteristically changed by arrest in their specific stage of cell division cycle without a notable alteration in the total cellular activity. The activities were low in the nuclei of cdc 25, cdc 28 and cdc 4, which were arrested in early to mid G1 phase by temperature shift-up, and in the nuclei of wild-type cells (A364A), which were arrested in early G1 phase by alpha-factor treatment, while high level of the activity was found in the nuclei of cdc 7 and cdc 8, which were arrested at late G1 and S phase, respectively. Activity-gel analysis of DNA polymerase in the nuclear extracts revealed the presence of two active peptides (120K and 72K), and the characteristic decrease in both active peptides was induced by arrest in early to mid G1 phase. Consequently, it is strongly suggested that intranuclear DNA polymerase activity alters in a dependent fashion on progression of cell division cycle. Subunit analysis indicated that the purified DNA polymerase I is constructed from two subunit peptides of 120K and 62K, and the large subunit possesses catalytic activity.     

Characterization of DNA polymerase alpha activity from a mouse DNA temperature-sensitive mutant, strain tsFT20, which shows a defect in DNA polymerase alpha activity at restrictive temperatures

tsFT20 cells derived from mouse FM3A cells are DNA temperature-sensitive mutants, which have heat-labile DNA polymerase alpha activity. When tsFT20 cells were incubated at restrictive temperatures, intracellular levels of DNA polymerase alpha activity changed biphasically, showing an initial fast decrease (phase I) and a subsequent slow decrease (phase II). The activity of DNA polymerase alpha from tsFT20 cells cultured at a permissive temperature (33 degrees C) was greatly increased by the addition of glycerol or ethylene glycol to the reaction mixture, while little increase in enzyme activity was observed at any concentration of glycerol or ethylene glycol tested with the enzyme from the cells cultured at a restrictive temperature (39 degrees C) for 8 h (phase II). The activity of DNA polymerase alpha from wild-type cells was also increased by the addition of glycerol but the increase was much less than that in the tsFT20 cells. An in vitro preincubation experiment showed that DNA polymerase alpha from tsFT20 cells cultured at 33 degrees C very rapidly lost its ability to be stimulated by glycerol. Furthermore, the experiment using the extracts prepared from tsFT20 cells cultured at 39 degrees C for various periods showed that the ability to be stimulated by glycerol decreased with the duration of incubation time at 39 degrees C. DNA polymerase alpha from the revertants, which can grow at 39 degrees C and exhibit a partial recovery in heat stability of DNA polymerase alpha activity, showed an intermediate response to glycerol, between those of DNA polymerase alpha from tsFT20 and from the wild-type cells. Finally, it was observed that the level of enzyme activity that can be stimulated by glycerol correlated well with the DNA synthesizing ability of tsFT20 cells.     

Serum-dependent regulation of proliferation of cultured rat fibroblasts in G1 and G2 phases

We reported that: (i) 3Y1tsF121 cells, a temperature-sensitive (ts) mutant of rat 3Y1 fibroblasts, are reversibly arrested either in the G1 or in the G2 phase, at the nonpermissive temperature. (ii) Cells retain the ability to resume proliferation at the permissive temperature after prolonged arrest in the G1 phase (for 5 days), whereas they lose it after prolonged arrest in the G2 phase (over 24 h). (iii) The G1 arrest is overcome at the nonpermissive temperature by the addition of fresh serum (H. Zaitsu and G. Kimura (1984) J. Cell. Physiol. 119, 82; (1985) J. Cell. Physiol. 124, 177). In the present study, the G2 arrest was overcome by exposing the cells to fresh serum, at the nonpermissive temperature. The G2 arrest occurred only at a higher cell density than that of the G1 arrest. The efficiency of the overcome was higher in the case of the G2 arrest than in case of the G1 arrest. When cells synchronized at the G1/S border by aphidicolin at the permissive temperature were released from the block, they divided in the absence of serum, at the permissive temperature. Even if they had passed through the previous G2 phase in a very high concentration of fresh serum at the permissive temperature, mitotic cells did not enter the S phase in the absence of serum, even at the permissive temperature. When the cells arrested in the G1 phase (not in G0) due to the ts defect were incubated in the absence of serum at the permissive temperature, only 34% entered the S phase and only 15% divided. These results suggest that (i) the ts defect in 3Y1tsF121 limiting cellular proliferation in both the G1 and the G2 phases is probably due to a single mutational event, and is a serum-requiring event. (ii) Preparation of the serum-requiring event which is required for the G2 traverse is completed in the G1 phase, under ordinary conditions. (iii) However, cells are able to fulfill the serum-requiring event in the G2 phase as well as in the G1 phase when the preparation is below the required level. (iv) The commitment to DNA synthesis is not necessarily a commitment to cell division. (v) Cells are arrested in the G1 phase more safely and more effectively than in the G2 phase, by the serum-related mechanism.     

Exposure of HeLa DNA polymerase alpha to protein kinase C affects its catalytic properties

Protein kinase C (Ca2+/phospholipid-dependent protein kinase) purified from rat brain or endogenous to cell-free extracts from HeLa cells stimulates, by a factor of 2-3, HeLa DNA polymerase alpha but not beta or gamma. Monoclonal antibody to the kinase prevents the stimulation, and monoclonal antibody to human DNA polymerase alpha neutralizes the enhanced activity. Reduced DNA polymerase alpha activity is obtained from noncycling HeLa cells and this activity has lower fidelity when copying synthetic primer-templates than that obtained from log phase cultures. After exposure to the kinase, the fidelities and activities of the polymerase from both sources increase by 2- to 3-fold. This improved accuracy is not accompanied by the appearance of triphosphatase or DNase activities. Exposure to the protein kinase reduces the Km for activated DNA and for poly(dA-dT) but not for dNTPs. Moreover, the Vmax for activated DNA but not for poly(dA-dT) is increased approximately 2- to 3-fold. These alterations suggest a role for protein phosphorylation in modulating DNA polymerase alpha.     

Interferon affects cell growth progression by modulating DNA polymerases activity

A multiparametric analysis of the effects of human recombinant interferon alpha type A on Daudi cells involving flow cytometry and in vitro analysis of alpha and beta DNA polymerase activities has been performed. Results have disclosed (within 60 min of interferon treatment) a decrease of alpha polymerase driven DNA synthesis persisting to at least 24 h, while beta polymerase was poorly affected. Moreover, after 24 h of interferon treatment, a reduction of BrdUrd incorporation per cell, assessed by flow cytometry, was observed suggesting that DNA synthesis in S phase cells is almost completely abolished. The analysis of the effect of interferon on the distribution of cell cycle phases indicated that the G1/S transition is not inhibited by the treatment. These results support the hypothesis that interferon generates a transient initiating signal which quickly reaches the nucleus and produces a rapid inhibition of alpha polymerase activity, leading finally to the slowing of cell cycle progression.     

Phosphoinositide-dependent kinase l (PDK1) haplo-insufficiency inhibits production of alpha/beta (alpha/beta) but not gamma delta (gamma/delta) T lymphocytes

In the present study, we have explored the impact of deleting a single allele of PDK1 in T cell progenitors on alpha/beta and gamma/delta T cell development. The data show that deleting a single allele of PDK1 allows differentiation of alpha/beta T cells but prevents their proliferative expansion in the thymus. Accordingly, mice with T cells that are haplo-insufficient for PDK1 have reduced numbers of thymocytes and alpha/beta peripheral T cells. T cell progenitors also give rise to gamma/delta T cells but in contrast to the loss of alpha/beta T cells in T-PDK1 null and haplo-insufficient mice, there were increased numbers of gamma/delta T cells. The production of alpha/beta T cells is dependent on the proliferative expansion of thymocytes and is determined by a balance between the frequency with which cells enter the proliferative phase of the cell cycle and rates of cell death. Herein, we show that PDK1 haplo-insufficient thymocytes have no defects in their ability to enter the cell cycle but show increased apoptosis. PDK1 thus plays a determining role in the development of alpha/beta T lymphocytes but does not limit gamma/delta T cell development.     

Prolongation of duration of G2 arrest delays and finally blocks entry into M phase in contrast to stable and reversible G1 arrest: study of a G1/G2 temperature-sensitive mutant of rat 3Y1 fibroblasts

Proliferation of 3Y1tsF121 cells was arrested in G1 and G2 phases after a shift up to 39.8 degrees C (restrictive temperature). Both arrests were reversible: after a shift down to 33.8 degrees C (permissive temperature), these cells effectively entered the next phases. However, the entry into M phase of the G2-arrested cells was delayed depending on the time in arrest. The G2-arrested cells finally became incapable of entering M phase with a prolonged incubation at 39.8 degrees C. Under the same condition, G1-arrested cells did not lose their ability to proliferate, and their delay of entry into S phase was slight. Therefore, cells in G2 phase are, in a sense, more unstable than the cells in G1 phase. These results also suggest that the time required for entry into M phase may depend on the preparedness for the initiation of M phase and, that it may be prolonged under the condition where the preparedness for entry into M phase is diminished.     

Calmodulin regulates DNA polymerase alpha activity during proliferative activation of NRK cells.

When Normal Rat Kidney cells are allowed to reenter the cell cycle after quiescence they start to replicate DNA around 12 h, reaching a maximum at 20 h. Activation of DNA polymerase alpha parallels the increase in DNA synthesis. The addition of two different anti-calmodulin drugs, trifluoroperazine (7.5 microM) or W13 (10 micrograms/ml), to the media at 4 h after proliferative activation, inhibits DNA synthesis by 55% and 80%, respectively. The blockade of calmodulin produced by trifluoroperazine allows the cells to progress through G1 phase but stops progression through S phase as determined by 5-Bromo deoxyuridine labeling. Both anti-calmodulin drugs also inhibit by more than 50% the increase in DNA polymerase alpha activity observed at 20 h. These results indicate that a calmodulin-dependent event, essential for the activation of DNA polymerase alpha and subsequently for DNA replication, is produced during G1. Therefore, the control of DNA polymerase alpha activation is one of the ways by which calmodulin is regulating the progression of NRK cells through S phase.     

Studies of Schizosaccharomyces pombe DNA polymerase alpha at different stages of the cell cycle.

The status of Schizosaccharomyces pombe (fission yeast) DNA polymerase alpha was investigated at different stages of the cell cycle. S.pombe DNA polymerase alpha is a phosphoprotein, with serine being the exclusive phosphoamino acid. By in vivo pulse labeling experiments DNA polymerase alpha was found to be phosphorylated to a 3-fold higher level in late S phase cells compared with cells in the G2 and M phases, but the steady-state level of phosphorylation did not vary significantly during the cell cycle. Tryptic phosphopeptide mapping demonstrated that the phosphorylation sites of DNA polymerase alpha from late S phase cells were not the same as that from G2/M phase cells. DNA polymerase alpha partially purified from G1/S cells had a different mobility in native gels from that from G2/M phase cells. The partially purified polymerase alpha from G1/S phase cells had a higher affinity for single-stranded DNA than that from G2/M phase cells. Despite the apparent differences in cell cycle-dependent phosphorylation, mobility in native gels and affinity for DNA, the in vitro enzymatic activity of the partially purified DNA polymerase alpha did not appear to vary during the cell cycle. The possible biological significance of these cell cycle-dependent characteristics of DNA polymerase alpha is discussed.