Nuclear Location of Mammalian DNA Polymerase Activities*

Nuclei were isolated from monolayer cultures of mouse and cells using a nonaqueous procedure of cell fractionation

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 for 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 halflives of 1 h at 23" and 4 h at O-4". Activities were relatively stable at -25", however, so that by homogenizing only 15 min at 4" and centrifuging at -25" 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-cu and -p in approximately the proportions expected if all polymerase activities were confined to the nucleus in living cells. DNA polymerase-a was found to be more unstable in glycerol suspensions than DNA polymerase+.
Nuclear location of both activities was found in exponential cultures and in 3T3 mouse cultures synchronized in the G, 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-cu, frequently is present as a weakly bound nuclear protein. -A more detailed description of this method will be published elsewhere (33). Monolayer cultures were washed with NaCl/Pi2 at 2" and trypsinized (100 pglml of trypsin in NaCl/P,) for 2 to 8 min at 2" (34). The trypsin solution was decanted, residual trypsin was inactivated by rinsing with 20 yglml of soybean trypsin inhibitor in NaCl/P,, and the tenuously attached monolayers were washed twice more with NaCl/P,. The cells were then suspended in NaCl/P, by pipetting and centrifuged (1000 x g, 30 s, 2"). The pellet of cells (~0.5 ml) was suspended in 2 or 3 times its volume in either NaCl/P, or in 10 rnM sodium phosphate (pH 7.2) and then frozen by dripping the concentrated cell suspension into melting Freon-12' (m.p. -158"). The Freon-12 was removed and a test tube containing frozen cells was evacuated for 12 to 36 h while being refrigerated at -28". When pressure near the cells reached 3 Torr, the dried cells were mixed with 2 to 4 ml of anhydrous glycerol at 2" and homogenized at 4000 to 6000 rpm for 15 min at O-4" using a Vir-Tis-23 microhomogenizer (Vir-Tis Co., Gardiner, N. Y.) with a single l-cm blade. The homogenized cells were poured into a centrifuge tube over a layer of glycerol and were centrifuged at 40,000 rpm (130,000 x g,,) for 12 to 18 h at -25". The nuclear pellet and cytoplasmic supernatant were then separated at 2" and stored at -28" or -76".
The extraction mixture was next mixed at 2" with an equal volume of the solution containing activated DNA primer-template described above. Triplicate samples of 100 ~1 were incubated for 5 min at 37". The reaction was stopped by adding 1 ml of ice-cold 10% (w/v) trichloroacetic acid plus 30 rnM Na,PP,. After 30 min at o", precipitated material was collected by suction on Whatman GF/C glass fiber filters. The filters were washed five times at 0" with 2 ml of N HCl plus 30 mM Na,PP, and twice with 95% ethanol.
Each filter was then air-dried and extracted for 5 min at 23" with 0.25 ml of NCS solubilizer (Amershamisearle) and mixed with 5 ml of toluene-based scintillation fluid (4.6 g of New England Nuclear Omnifluorlkg of toluene).
Samples were counted in a Nuclear Chicago scintillation counter at 50% efficiency.
In Table I

RESULTS
Cell Fractionation-As reported elsewhere (33) our nonaqueous procedure yielded nuclear and cytoplasmic cell fractions contaminated with approximately 10% of the RNA species characteristic of the opposite fraction. Cytoplasmic fractions contained 5 to 10% of cellular DNA. Electrophoresis of nuclear and cytoplasmic proteins also showed low levels of cross-contamination of the two cell fractions although the relative amounts were not quantitated (33).

Recovery of Polymerase Activity in Fractionation
-Four types of cells, 3T3-G,, 3T3-S, SVTB, and KB, were fractionated and the specific activity of DNA polymerases was measured in extracts made at seven steps during fractionation.
Results are shown in Table I. Specific activity .(polymerase activity divided by DNA content) was retained quantitatively through the point of mixing dried cells with glycerol. During homogenktion, approximately 15% of the specific polymerase activity was lost. During centrifugation, 0 to 3% of the total activity was also lost. Of the centrifuged homogenate, 90 to 95% of the DNA polymerase activity was recovered in the nuclear fraction, and 5 to 10% of the DNA polymerase activity was recovered in the cytoplasmic fraction. Isolated nuclei contained DNA polymerase-specific activities which were 83 to 89% of the specific activities of fresh whole cells. Because of the temperature-dependent inactivation of activity, we minimized exposure of lyophilized material to glycerol at temperatures above -25". Homogenization below 0" was impossible due to the high viscosity and occasional freezing of supercooled glycerol (m.p. +17X), but homogenization could be completed in 15 min at 0" followed by prompt chilling on solid COZ. After centrifugation (130,000 x g,,, 12 to 18 h, -25"), the cytoplasmic supernatant fraction was decanted at a temperature just warm enough to pour, approximately o", and then the separated fractions were stored at -28" or -76". The time of exposure of lyophilized material to glycerol above -25" was thus held to approximately 30 min. Losses of 50% or greater occurred if centrifugation was perfbrmed at higher temperatures (130,000 X g,,,, 2 h, 0"). Therefore, a loss of 11 to 17% in specific polymerase activity in isolated nuclei (Table I) could have been due to the temperature-dependent inactivation observed in Fig. 1.

Sedimentation
Analysis of Polymerase Activities -Sucrose   (Figs. 2 and 3). In extracts from mouse cells and nuclei, the 3.4 S species was more active at pH 8.6 than at pH 7.2, while the 7.0 S species was more active at pH 7.2. The 7.0 S activity, from mouse or human material, was abolished when incubated in 10 mMN-ethylmaleimide, while the 3.4 S activity was unaffected (not shown). These differences between the two activities allow us to identify the 7.0 S activity as DNA polymerase-a and the 3.4 S activity as DNA polymerase-P (1, 2).
The ratio of the two activities, (Y/P, each measured at its preferred pH ("Experimental Procedures"), was 1.6 to 2.7 in extracts from SVT2 cells or nuclei and 2.3 to 3.7 from asynchronously dividing 3T3 cells or nuclei (Fig. 2). The ratio obtained from nuclei was 60 to 100% of that obtained from whole cells. In an experiment described in Fig. 2C, the glycerol homogenate and nuclear pellet were kept above -25" for more time than usual in our procedure and the ratio obtained from isolated nuclei was 0.26. Although somewhat variable, these data confirmed nuclear location of both activities and also showed that the majority of the temperature-dependent inactivation of polymerase activity at O-4" was confined to DNA polymerase-cu.
Relative amounts of activities were also investigated in synchronized 3T3 cells and in nuclei isolated from them. The ratio, a/p, measured again at the two preferred pH values, was 2.3 to 3.8 in extracts from S phase material, 1.4 in 6-day G, material, and 0.4 to 0.5 in 7-day G, material. Again, values obtained from whole cells and from nuclei were similar. These data showed that the relative amounts of the two activities were similar in S phase cells and in rapidly dividing asynchronous cells and that the amount of DNA polymerase-a activity relative to DNA polymerase-P activity declined as cells rested in G, phase.  Sedimentation of activities extracted from whole SVT2 cells and from isolated nuclei. Extracts were prepared from whole frozen SVT2 cells (A) and isolated nuclei (B and C) and analyzed by sedimentation in 5 to 20% sucrose 0.9 M K+ for 24 h at +4". In B glycerol-containing suspensions were exposed to temperatures above -25" for approximately 30 min. In C glycerol suspensions were exposed to +4" for 3 h and +23" for 30 min. The arrows denote the sedimentation positions of fluoresceinated bovine serum albumin (4.3 S) and IgG (7.0 S). Sedimentation was from left to right. Fractions were assayed for DNA polymerase activity at pH 8.6 (0) and pH 7.2 (0). Activity is picomoles of dATP incorporated into acidinsoluble material in a 30-min incubation.
The cytoplasmic cell fraction, after correction for known 5 to 10% contamination by nuclear material, contained no more than 5% of the DNA polymerase activity of the cell. Hence, we have concluded that the two major DNA polymerase activities of mouse and human cells are confined to the nucleus, although one, DNA polymerase-a, can be eluted easily from nuclei in aqueous media. This finding confirms earlier reports (13-21, 23-26) on a more quantitative basis and disputes the widely held notion that DNA polymerase-a! of dividing cells is cytoplasmic (39).
Electron micrographs of nuclei isolated by our procedures showed typical nuclear morphology except that the outer nuclear membrane and associated ribosomes were largely missing (22, 331. Both polymerase activities are therefore probably located in the region bounded by the inner nuclear membrane and cannot be present in any appreciable amounts in the perinuclear region (23). The apparent physical associations of polymerase activities with cytoplasmic membranes or with other enzymatic activities (40,41) should be re-examined with nonaqueous fractionation.
The cellular location of DNA polymerase-cu was nuclear in contact-inhibited G, phase 3T3 cells as well as in serumstimulated S phase 3T3 cells and in rapidly dividing 3T3, SVT2, and KB cells (Table I). Nuclear location during all stages of the cell cycle argues against a proposed migration of cy toplasmic polymerases to the nucleus at the beginning of DNA synthesis (42)(43)(44)(45). Also, the nearly quantitative recovery of activity in the nuclear fraction argues against a significant contribution to polymerase activity from other cytoplasmic factors. It is possible that recently described factors which can enhance polymerase activity (46) are also weakly bound nuclear components. The example of one weakly bound nuclear protein of some interest, DNA polymerase-a, should promote re-examination of cellular locations of other macromolecules. Levels of extractable DNA polymerase activity, DNA polymerase-a in particular, have been observed to be higher in proliferating cells than in resting cells (9,11,(47)(48)(49). Baril and Laszlo (41) compared polymerase activities from several rat tissues and observed a rough proportionality between activity and rates of DNA synthesis. These observations supported the contention that the rate of DNA replication is controlled by the activity of DNA polymerase-a. In our work with synchronized 3T3 cells, however, we observed that S cultures contained approximately 1000 times as many cells synthesizing DNA as did G, cultures, hence a IOOO-fold higher rate, yet the amount of DNA polymerase-oc activity in G, cultures was lower than that in S cultures by only a factor of 2 to 5. These observations showed that in 3T3 cells the frequency of initiation of S phase and overall rate of DNA synthesis were not simply proportional to the extractable activity of DNA polymerase-cu.
Although polymerase activities are nuclear, the elution of DNA polymerase-a! activity from nuclei during aqueous cell fractionation might be evidence for an inactive storage form, within the nucleus, for the majority of DNA polymerase-cu molecules. Cells engaged in exceptionally rapid DNA synthesis, such as early cleavage embryos (15, 501, regenerating rat liver (10,14), or mouse cells productively infected with poly-oma virus (511, have been reported to have exceptionally high proportions of total DNA polymerase or DNA polymerase-a! tightly bound to nuclei after conventional aqueous fractionation. If DNA polymerase-a is directly involved with DNA replication, which is by no means certain, then levels of more tightly bound DNA polymerase-oc should be more nearly proportional to rates of DNA synthesis than levels of total DNA polymerase-cy. Experiments testing this prediction have not yet been done.