Search for multienzyme complexes of DNA precursor pathways in uninfected mammalian cells and in...

JOURNAL OF CELLULAR PHYSIOLOGY 134:25-36 (1988)

Search for Multienzyme Complexes of DNA Precursor Pathways in Uninfected Mammalian

Cells and in Cells Infected With Herpes Simplex Virus Type I

BY GlLLlAN HARVEY AND COLIN K. PEARSON* Department of Biochemistry, University of Aberdeen, Marischal College, Aberdeen,

AB9 ?AS, Scotland

Confirmatory evidence for the existence of a multienzyme complex of DNA precursor pathways in mammalian cells was obtained. Using neutral sucrose gradient centrifugation of cell lysates we found that at least five enzymes involved in DNA precursor metabolism in uninfected, S-phase BHK-cell fibroblasts cosediment at a common rate, indicative of a multienzyme complex. The enzymes include DNA polymerase thymidine kinase, ribonucleotide reductase, dihydrofolate reductase, and NDP-kinase. This complex was partially, but not completely, disrupted when lysates from G,-phase cells were centrifuged. Using lysates from cells infected with herpes simplex virus (HSV) type I some of the virus-induced ribonucleotide reductase and a minor proportion of the HSV-thymidine kinase cosedimented rapidly. The virus-induced DNA polymerase sedimented independently near the middle of the gradient, in contrast to the behaviour of the host polymerase. The enzyme associations observed were disrupted by NaCl or by inclusion of ethylenediamine tetra- acetic acid during the cell lysis procedure, instead of the usual EGTA. These results indicate the importance of ionic forces in maintaining the enzyme complexes. The bulk of the DNA and the RNA present in the lysates did not sediment at the same rate as the complexes, showing that the enzymes were not simply adhering nonspecifically to these polyanions. Newly synthesised radiolabeled DNA (15 min pulse with [3H]thymidine) was not preferentially associated with the enzymes, but some functional DNA was evident in the enzyme complex fraction from the uninfected S-phase cells. DNA polymerase activity in this fraction did not require, nor was it stimulated by, exogenous “activated” DNA. Added DNA primer-template was required, however, for maximal activity of the polymerase in gradient fractions derived from Go- phase cells and from HSV-infected cells. No evidence for channeling of ribonucleotide precursors into DNA of permeabilized cells (uninfected or HSV-infected) was detected. Most rCDP was incorporated into RNA. In the uninfected, S-phase cells about 10 pmol/106 cells/90 min of rCDP residues was incorporated into DNA compared with 120 pmol/106 cells /90 min when radiolabeled dCTP was used. Nonradioactive dCTP present in equimolar concentration in the incubation with labeled rCDP did not, however, diminish the incorporation of label from the ribonucleotide. In permeabilized HSV-infected cells incorporation of radiolabel from rCDP into DNA was barely detectable.

Much evidence has accumulated over the last 10 years to suggest that the enzymes of DNA precursor biosynthesis function as a multienzyme complex that physi- cally l inks the synthesis of deoxyribonucleotides and their uti l isation in DNA replication (reviewed by Ma- thews, 1985; Mathews and Slabaugh, 1986). Such a complex would serve to channel distal precursors through relevant enzyme-catalysed steps leading to concentra- t ion gradients of substrates, eventually supplying replication pools, which may wel l be highly concentrated at replication sites.

Most of the early evidence for the existence of multienzyme complexes of DNA precursor pathways and for 0 1988 ALAN R. LISS, INC.

precursor channeling originated from studies w i t h the bacteriophage T4-infected Escherichia coli cell. Reddy et al. (1977) described the cosedimentation of f ive phage- coded enzymes and one host enzyme known to be involved in DNA precursor metabolism upon sucrose density gradient centrifugation of lysates from virus-infected cells. Simi lar reports appeared from the laboratory of Tomich et al. (1974), and Flanegan and Greenberg (1977).

Received for publication May 29, 1987; accepted September 16, 1987. *To whom reprint requests/correspondence should be addressed.

HARVEY AND PEARSON 26

Subsequent work has now added to the number of enzymes considered to be part of such a complex (Allen et al., 1980, 1983; Chiu et al., 1982). In all, about ten enzymes involved in providing DNA precursors in phage- T4-directed DNA synthesis have been identified. Other experiments have indicated the preferential channeling of distal precursors into DNA, providing kinetic evidence for the expected functioning of such a complex (reviewed in Mathews, 1985).

Several theoretical advantages for organizing enzymes into a complex have been considered (Perham, 1975; Welch, 1977). These include, for example, the co- ordinated effects of having enzymes in close proximity, whereby conformational alterations, resulting from substrate or effector binding, can be directly transmitted to neighbouring enzyme molecules. Enzyme clusters may compartmentalize their substrates without the need for a physical barrier such as a membrane (Gaertner, 1978). This would enable substrates to be sequestered where they are required and at high concentration, important where the K, is high. Close proximity of the active sites might mean that the diffusion of solutes from one enzyme active site to another may no longer be a rate consideration, i.e., there could be a decrease in the transit time. Such a system might then enable a more rapid transition from one steady-state flux through the metabolic pathway to another under changing physio- logical circumstances. Such a system operating in DNA precursor metabolic pathways would then enable coor- dinated regulation of component enzymes, providing a tight coupling mechanism between DNA precursor sup- ply and demand, as well as perhaps explaining the tem- poral functioning of the enzymes during the cell cycle (Kornberg, 1980).

We began work with the herpes virus-infected mammalian cell because it appeared to be an analogous situation to the phage T4/E. coli system. Like the phage, herpes simplex virus (HSV-1) encodes a number of DNA precursor pathway enzymes in its genome, which are synthesised after infection of the host cell. These include a DNA polymerase (Keir and Gold, 1963), deoxypyrimidine kinase (Jamieson et al., 19741, and a ribonucleotide reductase (Cohen, 1972). Evidence available a t the time our work began indicated that DNA presursor pathway enzyme complexes probably did exist in mammalian cells @eddy and Pardee, 1980, 1982, 1983; Ayusawa et al., 1983; Noguchi et al., 1983; Wickremasinghe et al., 1982, 1983). Furthermore, Ayusawa et al. (1983) carried out a genetic experiment that established the specificity of the protein-protein interactions that they observed.

MATERIALS AND METHODS Cell culture, infection with HSV-1

The routine culture of BHK-21/C13 cells was as previously described (Baybutt et al., 1982). Confluent cultures were infected with virus (HSV-1, strain 17: Medical Re- search Council, Institute of Virology, Glasgow) at a mul- tiplicity of infection of 20.

Cell synchrony Cells were arrested in growth by serum deprivation

(0.25% (v/v) serum) for up to 4 days (Go-cells), with a medium change after the first 24 h. Cells in S-phase were obtained 16 h after replenishment of full growth medium.

Preparation of cell lysates Cells were suspended (1-3 x lo7 cells/ml) in hypotonic

buffer consisting of 20 mM Tris-HC1, pH 7.5 at 4"C, 1.0 mM dithiothreitol, 2 mM MgC12, and 3 mM CaC12 and allowed to swell for 25 min. The supension was then sonicated briefly, and after the addition of micrococcal nuclease (100 unitdm1 final concentration; from Staph- ylococcus aureus, Sigma) and phospholipase C (60 units/ ml, Sigma) it was incubated for 1 h at 15°C. Reactions were stopped by the addition of 0.2 M EGTA (40 pYm1 of lysate), and the suspension was centrifuged for 15 min at 15,OOOg,. The resulting supernatant was used as the cell lysate.

Sucrose density gradients Linear sucrose gradients were prepared by mixing 5%

(w/v) and 20% (w/v) sucrose solutions, both in 20 mM Tris-HC1, pH 7.5 at 4"C, 1.0 mM dithiothreitol, and 2 mM MgC12. The linear gradient was layered over a 0.4- ml cushion of 66% (w/v) sucrose in the hypotonic buffer. Samples (400 pl of cell lysate) were applied to the top of the gradients (six identical gradients per rotor), which were then centrifuged at 35,000 r.p.m. for 8 h at 4°C in a Beckman SW50.1 rotor. Fractions (0.5 ml) were collected by pumping the gradient from the bottom through a DADE micropipette (50 1.1). Refractive index measure- ments of fractions were routinely made to establish re- producible preparation of gradients. An enzyme unit is defined as 1 pmol of substrate utilized per 30 min.

Enzyme assays BHK-cell DNA polymerase. Under the conditions used

polymerase a was the predominant enzyme activity measured. Assays (200 pl) contained 50 mM Tris-HC1, pH 7.5 at 37"C, 5 mM mercaptoethanol, 10 mM KC1, 8 mM MgClz, 0.2 mM each of dATP, dCTP, and dGTP, 0.02 mM [ HIdTTP (sp. act. 100 mCi/mmol), and 40 pg of "activated" salmon sperm DNA (Loeb, 1969). Reac- tions were started by adding 50 p1 of gradient fraction (10-50 pg of protein) to 150 p1 of assay mix. Incubations were for 20 min at 37°C. They were then precipitated with ice-cold trichloroacetic acid onto filter discs and counted for radioactivity.

HSV-1 DNA polymerase. Assays (200 p1) contained 100 mM Tris-HC1, pH 8.2 at 37"C, 5 mM dithiothreitol, 100 mM KC1, 3 mM MgC12$0.26 mM each of dATP, dCTP, and dGTP, 0.02 mM [ HIdTTP (sp. act. 20 mCi/ mmol), and 20 pg of "activated" salmon sperm DNA. Reactions were started by addition of 50 p1 of sucrose gradient fraction. Incubation conditions and subsequent processing of samples was as described for the BHK-cell enzyme.

Thymidine kinase. (Jamieson and Subak-Sharpe, 1974). The conversion of [3H]TdR to dTMP was deter- mined in an assay (200 pl final volume) containing 100 pl of sample (20 to 100 pg of protein), 50 mM Tris-HC1, pH 8.0 at 37°C (for HSV-TK this was 20 mM potassium phosphate buffer, pH 6.01, 10 mM ATP, 10 mM MgC12, and 10 pM [3H]TdR (5 Ci/mmol).

Ribonucleotide reductase. For assaying the BHK-cell enzyme the method of Reddy and Pardee (1982) was used. The final assay volume was 160 pl, and reactions were initiated by addition of 60 p1 of sucrose gradient fraction (10-25 pg of protein). Final concentrations of components were 50 mM Hepes, pH 7.2, 8 mM dithio-

27 MULTIENZYME COMPLEXES OF DNA PRECURSOR PATHWAYS

threitol, 10 mM MgC12,6 mM ATP, 60 pM FeC13, 8 mM NaF, and 100 pM r3H] rCDP (0.1 pCi/nmol).

The procedure used for measuring the activity of the HSV-induced ribonucleotide reductase included a step to decrease the content of NDP-kinase (Spector and Av- erett, 19831, an enzyme capable of phosphorylating the nucleoside diphosphate substrate to nonsubstrate nucleoside triphosphate. All gradient fractions to be assayed were precipitated with ammonium sulphate (0.29 g/ml of lysate), and the precipitate was collected by centrifugation. The pellet was redissolved in a minimum volume of 20 mM Tris-HC1, pH 7.6 at 4"C, containing 2 mM MgC12 and dialysed against the same buffer for 2 h. Portions of the dialysate (60 pl) were added to a ribonucleotide reductase assay mix (150 p1 final volume) containing 50 mM Hepes, pH 7.2, 5.0 mM dithiothreitol, 2 mM MgC12, 4-5 mM ATP, and 0.1 mM [3H]rCDP (0.3 pCi/nmol).

All incubations (host and virus enzyme assays) were at 37°C for 20-30 min and were terminated by placing tubes in a boiling water bath for 2 min. Samples were then cooled on ice, and 10 p1 of carrier deoxyribonucleoside (0.2 pM) and 10 pl of potato apyrase (22 pg) were added. After a 15-min incubation at 37"C, 5 p1 of 0.5 M Tris-HC1, pH 8.5 at 37"C, and 0.004 units of alkaline phosphatase (in 5 p1) were added, and the incubation was continued for a further 15 min. Reactions were terminated by boiling for 2 min, and precipitated material was removed by centrifugation. Samples of the SU- pernatant (50 pl) were applied to Dowex-l-borat,e columns to measure the amount of deoxycytidine formed in the reductase assay (Steeper and Steuart, 1970).

NDP-kinase. (Nickerson and Wells, 1978). This assay linked the phosphorylation of dTDP to dTTP and the oxidation of NADH to NADf, which was then monitored spectrophotometrically at 340 nM at 30°C. Reac- tions (3 ml final volume) contained 83 mM triethanolamine, pH 7.5, 17 mM MgC12, 67 mM KC1, 1 mM PEP, 4.4 mM ATP, 0.2 mM NADH, 0.2 unitdml of pyruvate kinase, and 9.0 unitdm1 of lactate dehydrogenase. The contents were mixed and allowed to stand for 3 rnin at 30°C before the gradient fraction (various volumes up to about 150 p1) was added. After another 2-3 min to enable any nonspecific oxidation of NADH to occur, dTDP (to 0.7 mM) was then added, and the decrease in absorbance over 5-10 min was measured.

Dihydrofolate reductase. (Mathews et al., 1963). The oxidation of NADPH to NADP' when dihydrofolate was reduced to tetrahydrofolate was measured spectrophotometrically at 340 nm. Assay volumes were 1.5 ml and contained the sucrose gradient fraction (usually between 20 and 150 pl), 33 mM potassium phosphate buffer, pH 7.5, 6.7 mM 8-mercaptoethanol, 67 pM NADPH, and 67 pM dihydrofolate. Incubations were for 10-15 rnin at room temperature.

Cell permeabilization with lysolecithin. (Miller et al., 1978, 1979). Five-centimeter dishes were seeded with about 0.5 x lo6 cells, which were allowed to grow for 24 h. Each dish was then washed three times with 3 ml of phosphate-buffered saline (PBS) followed by a final rinse in 3 ml of solution A (35 mM Hepes pH 7.4, 50 mM sucrose, 80 mM KC1, 4 mM MgC12, 7.5 mM potassium phosphate buffer, pH 7.4, and 1 mM CaC12). The required amount of lysolecithin (see Fin. 8) in 2 ml of

4°C. This solution was then immediately removed, and the cells, now permeable, were washed with 3 ml of solution A to remove remaining traces of lysolecithin.

When virus-infected cells were to be used, the cells were infected with HSV-1 8 h after plating. Cells were thus permeabilized 16 h after infection.

Incorporation of radiolabeled nucleotides into DNA of permeabilized cells. The conditions for optimum incorporation of radiolabel from rCDP and dCTP were different and were as follows: After washing permeabilized cells with solution A, 1 ml of incubation mixture containing the appropriate radioisotope was added to the dishes, which were then incubated at 37°C. For dCTP incorporation (or dTTP), the mix (Reddy and Par- dee, 1980) consisted of 55 mM Hepes, pH 7.4, 50 mM sucrose, 80 mM KC1, 4 mM MgC12, 7.5 mM potassium phosphate (pH 7.4), 1.0 mM CaC12, 10 mM phosphoenol- pyruvate, 1.25 mM ATP, 0.12 mM each of CTP, UTP, and GTP, and 0.2 mM each of dATP, dGTP, dTTP, and [3H]dCTP (0.1 Ci/mmol). For rCDP incorporation the components (Reddy and Pardee, 1982) were 50 mM Hepes, pH 7.4, 10 mM MgC12, 8 mM dithiothreitol, 60 pM FeC13, 0.75 mM CaC12, 10 mM phosphoenolpyru- vate, 3 mM ATP, 33 pM each of rCTP and rGTP, and 0.2 mM each of rGDP, rADP, dTDP, and [3H]rCDP (0.1 Ci/ mmol).

Reactions were terminated, after removing the assay solution and washing the cells, by addition of 3 ml of ice- cold 5% (w/v) trichloroacetic acid, 1% (w/v) pyrophos- phate. After 30 min at 4"C, precipitates from each dish were scraped into Eppendorf tubes and pelleted by bench centrifugation. The pellets were then each suspended in 0.75 ml of 0.4 M NaOH and incubated at 37°C for 2 h after which time a further 0.75 ml of the acid solution was added to precipitate nonalkali labile nucleic acid. After 30 min at 4"C, the suspensions were washed onto GFC filters for radioactivity determination.

Control experiments in which cellular RNA was previously labeled with [BHIuridine showed that at least a 1-h incubation with 0.4 M NaOH was required to decrease acid-insoluble radioactivity to background levels.

RESULTS Sucrose density gradient analysis of lysates from

uninfected cells

Figure 1 shows the centrifugation profile for five DNA precursor synthesizing enzymes using a lysate from cells in the S-phase of the cell cycle. Successful cell synchrony by the serum-deprivation method used was monitored by measuring radiolabeled TdR uptake into DNA in 10- min pulses and by measuring specific activities of DNA polymerase and thymidine kinase at intervals after addition of serum to the growth medium (details not shown). The major peak of activity is clearly in the same position in the gradient for all five enzymes, suggesting a cosedimentation of these molecules. Minor levels of activity of four of the enzymes appear nearer to the top of the gradient, where the bulk of the protein is present.

The situation when lysates from resting Go-phase cells were used is somewhat different (Fig. 2). Whilst DNA Dolvmerase. thvmidine kinase. and a portion of the ri- bok leo t ide reductase still c6sedime;ted with a rate

solution A was added to the plates andleft for 2 min at comparable to that of their S-phase counterparts, the

HARVEY AND PEARSON

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Fig. 1. Sucrose density gradient analysis of enzyme activities in lysates prepared from uninfected, S-phase BHK fibroblasts. Sedimenta- tion was from right to left. The sedimentation profiles shown are representative of six gradients centrifuged in the same rotor. Fractions were assayed for protein and enzyme activities either immediately or

after storage at -20°C. The arrows show the sedimentation of marker proteins. These were (from right to left) bovine serum albumin (66K), lactate dehydrogenase (146K), catalase (232K), and thyroglobulin (669K). An enzyme unit is defined as 1 pmol of substrate utilized per 30 min.

NDP-kinase and the majority of the dihydrofolate reductase were now sedimenting more slowly. The total protein distribution in these gradients was similar to that seen when the S-phase lysates were centrifuged.

It appears then that the previous association of all five enzymes is disrupted, although not completely abol- ished, when the cells are no longer in S-phase. The scales for enzyme activities on the ordinates of Figures 1 and 2 reflect the position of the cells in the cycle.

Sucrose gradient analysis of lysates from HSV-infected cells

Lysates were made from cells 16 h after infection with the virus. Maximum induction of virus-encoded enzyme activities occurred about this time, with activities being some 20- to 25-fold greater than activities present in the cells at the time of the initial infection. The rate of virus DNA synthesis was also maximal at 16 h postinfection.

Figure 3 shows that some of the HSV-induced ribonucleotide reductase and a small portion of the thymidine kinase rapidly cosedimented under these conditions (closed circles in Fig. 3, fraction 1-3). Most of the TK and some 40% of the RR sediment more slowly, where the bulk of the protein is found. Notably, and in marked contrast to the situation in the uninfected cell, the peak of the virus-induced DNA polymerase activity is inde-

pendent of these two enzymes (fraction 6). The total protein distribution is similar to that seen when lysates of uninfected cells are centrifuged.

Specific activities are shown because they amplify the changes in sedimentation behaviour of the rapidly sedimenting components (fractions 1-3) of the TK and RR with and without EDTA in the cell lysates (see text below and Fig. 4b).

Stability of enzyme associations seen in the sucrose gradients

In agreement with previous observations of Reddy and Pardee (1980) we found that exposure to 2 M NaCl disrupted the cosedimentation of the enzymes from the uninfected S-phase cells (this experiment was not carried out with virus-infected cells). We also observed a clear disruption of cosedimenting enzymes from both the uninfected and HSV-infected cells when EDTA was used to terminate the enzyme digest step during the preparation of lysates prior to gradient centrifugation (Fig. 4). EGTA was normally used for this purpose by sequester- ing required Ca2+ ions.

The figure shows that the two enzymes from the uninfected cells that were assayed (DNA polymerase and thymidine kinase) sedimented at much reduced rates (compare Fig. 1). In the HSV-infected cell situation, the

MULTIENZYME COMPLEXES OF DNA PRECURSOR PATHWAYS 29

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Fig. 2. Sucrose density gradient analysis of lysates from uninfected Go-phase cells. Conditions were as described for Figure 1.

TABLE 1. Approximate molecular weights of some mammalian cell DNA precursor pathway enzymes

Mammalian enzymes Molecular weight Reference

DNA polymerase 110-185K (catalytic subunit) Hiibscher (1984) Thymidine kinase 83-91K Kit et al. (1973) Ribonucleotide reductase 84K (principal polypeptide); Engstrom et al. (1979)

Dihydrofolate reductase 20-23K Nichol(1968) Nucleoside diphosphokinase 80-100K Cheng et al. (1971) HSV-1-induced enzymes

55K subunit (200-300K holoenzyme)

DNA polymerase 150K Weissbach et al. (1973) Thymidine kinase 74K Leung et al. (1975) Ribonucleotide reductase 144K and 38K (subunits) Bacchetti et al. (1984)

previously rapidly sedimenting TK component was no longer seen, and the proportion of the RR that sedimented rapidly was diminished (fractions 1-3). The changes in sedimentation behaviour of these two enzymes caused by EDTA is more clearly seen when specific activities are plotted (compare Figs. 3 and 4b, open circles). This is particularly noticeable with the TK where only a minor portion of the enzyme molecules sediments rapidly in the first instance (i.e., without EDTA). The peak of HSV DNA polymerase activity was unaffected by EDTA, supporting our previous observa- tion that this enzyme was not associated with the other virus-induced enzymes.

It seems apparent from the considerable effect of the EDTA on the sedimentation behaviour of most of the enzymes that divalent cation, possibly M 2 + , is important in maintaining the association normally seen between the enzymes.

Further characterisation of the enzyme complexes A number of considerations prompted the experiments

described below. The associations observed between the enzymes might simply represent artifacts of the cell lysis and gradient procedures. For example, we considered whether the enzymes were adhering ionically to the polyanions DNA andlor RNA. An association with some DNA, perhaps at the replication fork, might be expected for a complex of DNA precursor pathway enzymes, and this was examined also. Do the observed enzyme complexes contain template

DNA? DNA polymerase assays were carried out with or with-

out added primer-template DNA on appropriate gradient fractions. It is clear that the polymerase-containing fractions from uninfected S-phase cells, representing the complex of enzymes, already contain sufficient DNA for

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Fig. 3. Sucrose density gradient analysis of virus-induced enzyme activities in lysates prepared from HSV-1-infected BHK cells. Lysates were prepared 16 h after infection. Key: Enzyme activities (01, enzyme-specific activities (0).

activity to be measured (Fig. 5a). No further increase in activity was observed when DNA was added to the assay. It is tempting to consider that the integral DNA was being replicated at the time of the lysate preparation.

Results obtained from nonreplicating cells are more difficult to interpret because of the lower polymerase activity and also because this no longer sedimented as a distinct peak of activity. However, the addition of exogenous template DNA in this case did result in a detectable increase in polymerase activity (Fig. 5b).

When gradient fractions containing the HSV-induced DNA polymerase were considered little enzyme activity was measurable unless DNA was added to the assay (Fig. 5c,d). This is clearly different from the situation with the uninfected, S-phase cells. It should be noted that even with added DNA template there was no de-

Fig. 4. Effect of EDTA on the stability of enzyme associations during sucrose gradient centrifugation. During the preparation of cell lysates 40 111 of 0.2M EDTA was used to terminate the actions of the micrococcal nuclease and phospholipase C, instead of the usual EGTA. The figure shows sucrose gradient analysis of lysates from (a) S-phase BHK cells and (b) HSV-infected cells. In b the activities shown refer to the virus-induced enzymes. Key: Enzyme activities (O), enzyme-specific activities (0).

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MULTIENZYME COMPLEXES OF DNA PRECURSOR PATHWAYS 31 I I

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Fig. 5. Effect of exogenous DNA primer-template on the activity of DNA polymerase from sucrose gradients. The preparation of cell lysates and conditions of gradient analysis were as described in Materials and Methods. Gradient fractions were analysed for appropriate DNA polymerase activity either with (0) or without (0) added “activated” DNA. Lysates were from S-phase BHK cells (a), Go-phase cells (b), or HSV-infected cells (c) and (d).

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tectable DNA synthesis in the gradient fractions containing the cosedimenting HSV-TK and HSV-RR (fractions 1-3, Fig. 3).

Is newly replicated DNA associated with the cosedimenting enzymes?

Bulk DNA was long-labeled with [14C]TdR, and newly synthesized DNA was labeled by pulse-labeling for 15 min with r3H]TdR (Noguchi et al., 1983). Gradient analysis of lysates prepared on completion of the pulse-label was carried out under conditions identical with those used routinely for analysing enzyme activities.

When lysates from uninfected, S-phase cells were used, three components of DNA were observed in the gradients (Fig. 6a). The 3H and 14C profiles were similar. Little of the DNA, however, cosedimented with the complex of enzymes; the peak of enzyme activities routinely

Fig. 6. Sucrose density gradient behaviour of long-labeled and pulse- labeled DNA in cell lysates. The preparation of lysates and conditions of centrifugation were the same as those described for analysis of enzyme activities. After centrifugation, fractions, collected from the bottom, were precipitated with trichloroacetic acid onto filter discs and counted for radioactivity. a: Cells were serum-arrested and then allowed to progress into S-phase by replenishment of full growth medium containing 50 nCi/ml of [14C]thymidine (sp. act. 51 mCi/mmol). After 16 h the medium was removed, and the cells were pulse-labeled with [3H]thymidine (10 pCUml sp. act. 25 Cilmmol) for 15 min. b Cells were infected with HSV-1 in medium that contained [14C]thymidine (50 nCi/ ml, sp. act. 51 mCi/mmol). After 16 h the medium was exchanged, and the cells were pulse-labeled with [3H]thymidine (10 pCi/ml; sp. act. 25 Ci/mmol) for 15 min. Cells were then washed, and lysates were prepared for centrifugation. Key: I4C long-labeled DNA (O), 3H-pulse- labeled DNA (0).

HARVEY AND PEARSON 32

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Fraction Number

Fig. 7. Sedimentation behaviour of RNA in cell lysates. a: Serum- arrested cells were stimulated to grow by replenishment of full growth medium containing [3H]uridine (0.1 pCi/ml; sp. act. 20 Cilmmol). Cell lysates were prepared 16 h later and centrifuged in the sucrose density gradients (0). Cells maintained in the Go-phase by serum deprivation were similarly incubated with the radiolabeled uridine for 16h (0). b: Cells were infected with HSV-1 in medium that contained 0.1 pCi/ml of the [3H]uridine. After 16 h, cell lysates were prepared and centrifuged in sucrose gradients. Conditions of centrifugation and treatment of gradient fractions was as described for Figure 6.

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Lysolecithin (yg12ml)

Fig. 8. a: Effect of lysolecithin concentration on cell permeability and viability. Exponentially growing cells (about 0.8 x 106/dish) in 5cm- diameter dishes were permeabilized for 2 min using the different lysolecithin concentrations shown (abscissa as for part b). The permeabilizing medium was then exchanged for cell growth medium, and the cells were incubated at 37°C for 24 h. Control cells were incubated with permeabilizing solution without lysolecithin and then with growth medium for 24 h. Results are plotted as relative growth rate (%), that is, the ratio (as a percentage) of the permeabilized cell number, cor- rected for an 8% loss after permeabilization, to the control cell number after the 24-h incubation in growth medium (0). Expermimental points are the means k S.D. (n = 3). Cell permeability (%) was estimated from the uptake of dye from a 0.1% solution of Trypan blue (0). b: Effect of lysolecithin concentration on the uptake of radiolabel from dTTP into DNA. Exponentially growing BHK cells were permeabilized with the different concentrations of lysolecithin shown and then incubated for 30 min at 37°C in assay mix containing t3H]dTTP (see “Materials and Methods” for details). The results show incorporation of radioactivity into acid-insoluble material (means & range, n = 3).

appeared in the bottom half of the gradient, equivalent to about fraction 16-20 in Figure 6a (see Fig. 1). It is thus unlikely that the cosedimenting enzymes are simply adhering to a mass of DNA to create an artifactually fast sedimenting structure.

A preferential enrichment of replicating DNA at- tached to the enzyme complex is not apparent from these experiments.

In a similar experiment with lysates from HSV-infected cells there was a much broader distribution of both pulse-labeled and long-labeled DNA throughout the gradient (Fig. 6b). It is apparent from this that gradient fractions containing the virus-induced enzymes (see Fig. 3) would also contain some DNA. It is perhaps surprising, therefore, that exogenous DNA primer-template was required to detect the majority of the DNA Dolvmerase in this case.

Do enzymes cosediment with RNA? Figure 7a shows that little, if any, RNA was associated

with the cosedimenting enzymes from the uninfected cells. However, it is possible that the virus-induced RR and TK could be associated with some RNA, since they sediment near the bottom of the gradient under the conditions used (compare Fig. 3 with Fig. 7b).

Are ribonucleotides preferentially channeled into DNA?

Use of permeabilized cells. Previous studies had indicated that channeling of distal DNA precursors occurs, using isolated multienzyme complexes from phage T4- infected E. coli and some mammalian cell lines, and in Dermeabilized mammalian cells (Reddv and Pardee.

I d i982; Mathews, 1985; Noguchi et al., 1583; Wickrema:

33

to be about 20%. Kinetic experiments. Using this permeabilized cell

system, we observed no evidence for the preferential incorporation of ribonucleotides into DNA compared with deoxyribonucleotides, with either uninfected or HSV-infected cells (Fig. 9 a,b). The little radioactivity that is incorporated from the rCDP was shown to be DNA by its susceptibility to DNAase but not RNAase; these simple experiments do not enable us to discount a DNA-RNA copolymer such as that described by Reddy et al. (1986). Much of this, if formed, would probably have been hydrolyzed by our alkaline conditions. The addition of nonradioactive dCTP did not appear to decrease the incorporation of label from the ribonucleotide, which supported the notion of separate pools for these nucleotides, although the level of radioactivity incorporated from rCDP is too low to conclude this with any certainty.

DISCUSSION

MULTIENZYME COMPLEXES OF DNA PRECURSOR PATHWAYS

30 60 90

l n c u b a t i o n l i m e tmin)

Fig. 9. Comparison of the relative incorporation of ribonucleotides and deoxyribonucleotides into DNA in permeabilized cells. Exponen- tially growing BHK cells (a) or HSV-infected cells (b) on 5-cm dishes were permeabilized for 2 min and then incubated at 37°C in the appropriate assay mix (see Materials and Methods) containing the radiolabeled nucleotides. Key: Incorporation of radiolabel from r3H] dCTP into acid-insoluble radioactivity (O), from [3H]rCDP (A), or from [3H]rCDP + 0.2 mM nonradioactive dCTP (A).

singhe et al., 1982, 1983). We have used a cell system permeabilized with lysolecithin to investigate this (Miller et al., 1978, 1979).

The optimum conditions for such a system must en- sure that the majority of the cells are rendered permeable to exogenous molecules but that they are not so damaged as to be rendered nonfunctional. Figure 8a indicates that a lysolecithin concentration of about 100 pglml was optimal for these criteria. This was also the concentration at which cells exhibited maximum ability to incorporate radiolabel from dTTP into DNA (Fig. 8b). Other experiments (not shown) to characterise the system established that the cells permeabilized in this way incorporated label for up to 90 min, the rate being linear for about 30 min. The DNA synthesis taking place was shown to be replicative by comparing the kinetics of radiolabel incorporated into DNA from d l T P in permeabilized cells with that from TdR in intact, nonpermea- bilized cells at intervals after release from a low-serum block (for the noninfected cells) or at various times after infection with the HSV. Kinetics of incorporation of label from the different isotopes was similar in each case. Loss of protein from permeabilized cells was estimated

In view of the possible loose association of enzymes in a putative complex we spent a considerable time on ascertaining the simplest optimum conditions for lysing cells such that most of the DNA precursor enzymes could be obtained. After simple swelling in hypotonic medium followed by sonic disruption and removal of cell organelles by centrifugation at 30,000gav much of the enzyme activity remained in the pellet fraction. The inclusion of the DNAase and phospholipase C digestion (Wickremasinghe et al., 1982, 1983) was effective at releasing bound enzyme from nuclei and enabled quan- titative recoveries of enzymes in a subsequent supernatant fraction.

Density gradient analysis provided clear physical evidence for a multienzyme association in lysates from uninfected, S-phase cells (Fig. 1). For each of the five enzymes assayed, the highest activities and specific activities resided in gradient fractions containing the complex, although smaller peaks of activities appeared near the top of the gradient. This contrasts quantitatively with results presented by Reddy and Pardee (19801, in which the highest specific activities of two of the enzymes in a complex, DHFR and NDP-kinase, were in a different position in the gradient from the complex-containing fractions. Our use of whole-cell lysates rather than the nuclear lysates used by Reddy and Pardee may explain this difference. Further comparisons cannot be drawn since these workers plotted specific enzyme activities only, not total activity, and did not show the protein distribution in the sucrose gradients.

The molecular size of our complex can be estimated from reference to marker proteins in the sucrose gradients to be in the region of 700K. If the five enzymes assayed formed a single complex, and this is not certain, then it is unlikely that yet other enzymes may also have been present (see Table 1). Thus, the four enzymes of dNTP biosynthesis have an aggregate molecular weight of about 450K. If the complex were also to contain addi- tional enzymes such as thymidylate synthase, thymidylate kinase, dCMP deaminase, etc., there would not be room for replication proteins. It is possible, therefore, that we have detected a dNTP-synthesizing complex but not one that funnels precursors to DNA. If this is the case the co-sedimentation of the DNA polymerase may

34 HARVEY AND PEARSON

be fortuitous. In addition, the presence of most Of the NDP-kinase in the complex is puzzling, since this is quantitatively more important in ribonucleotide than in deoxyribonucleotide biosynthesis. The rapid sedimentation of most of this enzyme may, therefore, be unrelated to its role in dNTP biosynthesis.

Other estimates of comparable enzyme complexes include the report by Noguchi et al. (1983) of a “replitase” complex from mammalian cells of approximately 5 x lo6. More recently, Jackson and Cook (1986) have described a number of structures from HeLa cells containing DNA polymerase activity, including active complexes of 750K and 3 x lo6 in addition to enzyme activity tightly associated with the nuclear skeleton.

In view of the number of DNA precursor enzymes involved in the observed complexes, the question of intracellular location becomes important. At least three of the enzymes, NDP-kinase, DNA polymerase, and TK, were seen to reside in the nucleus during DNA synthesis (Reddy and Pardee, 1980). However, RR, established by us and previously by Reddy and Pardee (1982) as belong- ing to the complex of enzymes, appears to have a cyto- solic location (Engstrom et al., 1984; Youdale et al., 1984; Leeds et al., 1985; Kucera and Paulus, 1986; these latter authors also localized thymidylate synthase to the cytoplasm). Youdale et al. (1984) suggest that the holoenzyme, assembled in the cytosol, may be suddenly delivered to the nucleus where it becomes locked into a multienzyme complex. It can only be supposed at this time that any multienzyme system residing in the nucleus, and channeling distal ribonucleotide precursors into DNA, would require a nuclear site for RR.

We did not observe a clear demonstration of enzyme associations using the HSV-infected cell. Only two of the virus-induced enzymes, RR and TK, cosediment rapidly, and only a minor proportion of the TK was involved (less than 10%). Rapid sedimentation of the RR (2.5 times faster than the host cell enzyme) in glycerol gradients was previously reported by Huszar et al. (1983). How- ever, the enzyme was not associated with the other virus-induced enzymes DNA polymerase, thymidine kinase, and alkaline DNAase. It is possible that this was a consequence of the (NH&S04 fractionation used prior to centrifugation.

The HSV-induced DNA polymerase sedimented independently in the gradients. It does not even appear to be associated with bound DNA, which it could use as a primer-template, as seen for the host polymerase in the uninfected cell. We note, however, that it still sediments halfway down the gradient under the conditions used, i.e., at the same rate as the catalase reference protein of mol. wt. 232K. This is a faster rate than expected of an uncomplexed DNA polymerase molecule.

It is possible that only a loose association of the DNA polymerase with RR and TK exists, or that the polymerase is associated with other enzymes and proteins that were not assayed. Other reports have shown, for example, the copurification of both DNA-binding proteins and topoisomerase with HSV-1-induced DNA polymerase (Biswal et al., 1983; Vaughan et al., 1985). Thus, in the virus-infected cell the production and utilisation of DNA precursors might not be tightly coupled or may not even be coupled at all, a possibility discussed above also for the uninfected cell. In this context we note that Sla- baugh et al. (1984) failed to find an association between

ribonucleotide reductase and the DNA replication ap- paratus in another viral system, vaccinia.

It is interesting that the TK sediments as two separate components, the slower one with the bulk of the protein in the gradient (Fig. 3). It has been established that there is a considerable increase in cellular dTTP content following HSV-infection, seemingly in excess of that required for DNA synthesis (Jamieson and Bjursell, 1976a). It is possible that much of the TK producing this excess dTTP is not directly involved in providing a substrate for DNA polymerase and is not, therefore, associated into a precursor synthesizing complex. Its role may be to provide an increased pool of dTTP, outside a putative replication pool, which serves a different purpose from that of a DNA precursor, perhaps as a regulator molecule of the DNA replication process (Jamieson and Bjursell, 1976a,b; Ponce de Leon et al., 1977; Langelier et al., 1978).

In an analogous situation, Noguchi et al. (1983) showed that methotrexate-resistant CHEF-18 cells, which over- produce dihydrofolate reductase some 12-fold, exhibited only a two- to threefold increase in dhfr activity in the replitase fraction (multienzyme complex). This was taken as further evidence that the enzyme association was not a nonspecific aggregate of proteins.

Previous reports indicate that enzyme complexes in both prokaryotes and eukaryotes are sensitive to ionic strength changes (Reddy and Pardee, 1980; Chiu et al., 1982; Wickremasinghe et al., 19831, and our observations are in agreement with these, pointing to the importance of electrostatic forces in maintaining complex integrity. Our finding that enzyme cosedimentation in both the uninfected and the HSV-infected cell situation was disrupted when EDTA was used instead of EGTA in the cell lysis procedure (Fig. 4) also supports this notion. This is in agreement with results of Allen et al. (1980), who reported disruption of the T4 bacteriophage enzyme complex with millimolar amounts of EDTA but not EGTA. The greater a i n i t y of the EDTA for M 2 ’ ions suggests that this ion may be important in stabilizing the enzyme complex.

Both DNA and RNA can form complexes with metal ions. Any effect on metal ion binding to these polyanions might therefore be expected to influence their sedimentation behaviour and also that of any bound enzymes or other proteins. Since our observed enzyme complexes could have resulted from a non-specific electrostatic association with these polynucleotides, their disruption by 2 M NaCl or by EDTA is easily envisaged. However, radiolabeled DNA and RNA were shown to sediment in the sucrose gradients at rates different from those of the observed complex of enzymes, particularly from the uninfected cell (Fig. l), although the rapidly, cosedimenting RR and TK from the HSV-infected cells could have contained some DNA and RNA (Fig. 3).

It has previously been demonstrated that nascent DNA associates with the complex of DNA precursor enzymes in nuclear lysates of S-phase mammalian cells (Noguchi et al., 1983). Although we did not observe this under comparable labeling conditions (i.e., a 15-min pulse with [3H]TdR), we did establish that the enzyme complex from the uninfected S-phase cells does contain bound DNA, which the DNA polymerase component utilizes as a primer-template in an in vitro assay (Fig. 5a). Addition of exogenous DNA to the assay did not result in addi-

MULTIENZYME COMPLEXES OF DNA PRECURSOR PATHWAYS 35 tional DNA synthesis, as it did when gradient fractions Baybutt, H.N., Murray, B.A., and Pearson, C.K. (1982) Aspects of from Go-phase cells, or from HSV-infected cells, were thymidine metabolism and function in cultured mammalian cells used. infected with herpes simplex virus type I. J. Gen. Virol., 59:223-234.

Biswal, N, Feldan, P., and Levy, C.C. (1983) A DNA topoisomerase

bonucleotide precursors into DNA was obtained using plex virus. Biochim. Biophys. Acta, 740:397-389. uninfected cells or HSV-infected cells rendered perme- Chew, y-c. , Agarwal, R.P., and Parks, R.E. Jr. (1971) Erythrocyte able to exogenous nucleotides, although this has previ- nucleoside diphosphokinase. W. Evidence for electrophoretic hetero-

geneity. Biochemistry, 20:2139-2143.

and Pardee, 1982, 1983). Indeed, no significant incorpo- a bacteriophage T4-induced complex synthesizing deoxyribonucleo- ration of label into DNA of HSV-infected cells was mea- tides. J. Biol. Chem., 257:15087-15097. surable. Most of the radiolabel in both situations was Cohen, G.H. (1972) Ribonucleotide reductase activity of synchronized

KB cells infected with herpes simplex virus. J. Virol. 9:408418.

by our NaOH digestion conditions, which were more Ribonucleotide reductase from calf thymus. Purification and proper- rigorous than those described by Reddy et al. (1982) in ties. Biochemistry, 18:2941-2948. view of the criticism of these by spyrou and Reichard Engstrom, y., Rozell, B., Hansson, H.-A., &emme, s., and Thelander,

L. (1984) Localization of ribonucleotide reductase in mammalian (1983). The small amount of label from rCDP incorpo- cells. EMBO J., 3:863-867. rated into DNA in the uninfected, S-phase cells (about Flanegan, J.B., and Greenberg, G.R. (1977) Regulation of deoxyribo- 10 pmoV106 cells in 90 min) was much less than that nucleotide biosynthesis during in uiuo bacteriophage T4 DNA repli-

cation. J. Biol. Chem., 252:3019-3027. from dCTP (120 pmo'106 ce11s'90 and thus provides Gaertner, F.H. (1978) Unique catalytic properties of enzyme clusters. no supporting evidence for the preferential hxorpora- Trends Biochem. Sci., 3:63-65. tion of the more distal precursor. This conclusion as- Hiibscher, U. (1984) DNA polymerase holoenzymes. Trends Biochem. sumes similar intracellular specific radioactivities of the si .7 9:390-393. different isotopes dictated by the experimental con- Huszar, D., Beharry, S., and Bacchetti, S. (1983) Herpes simplex virus-

induced ribonucleotide reductase: Development of antibodies specific ditions. for the enzyme. J. Gen. Virol., 64:1327-1335.

Our experiments in summary do identify associations Jackson, D.A., and Cook, P.R. (1986) Different populations of DNA of a number of DNA precursor enzymes, most notably in Po!Ymerase in HeLa cells. J. Mol. Biol., 192:77-86. the uninfected S-phase cells, which are unlikely to be Jamieson, AT., and Subak-Sharpe, J.H. (1974) Biochemical studies on

the herpes simplex virus-specified deoxypyrimidine kinase activity. artifacts of the experimental procedures used for reasons J. h n . viral., ~ 4 8 1 - 4 9 2 . discussed above. we were unable to demonstrate the Jamieson, A.T., and Bjursell, G. (1976a) Deoxyribonucleoside triphos- anticipated functional characteristics of such a complex Phate Pools in herpes simplex type I infected cells. J. Gen. VirOlv (i.e., substrate channeling), it is possible that Jamieson, A.T., and Bjwsell, G. (1976b) Deoxyribonucleoside triphos- the permeabilized Cell system is unsuitable for this Pur- phate pools in cells infected with deoxypyrimidine kinaseless herpes pose. For example, Pawlack et al. (1986) recently re- simplex virus. J. Gen. Virol., 31:115-123.

that permeabilized mammalian cells may not Jamieson, A.T., Gentry, G.A., and Subak-Sharpe, J.H. (1974) Induction provide a true representation of DN~-template-depen- of both thymidine and deoxycytidine kinase activity by herpes vi-

ruses. J. Gen. Virol., 24t465-480. dent nucleic acid synthesis. Keir, H.M., and Gold, E. (1963) Deoxyribonucleic acid nucleotidyltrans-

A most intriguing feature with the HSV-infected cells ferase and deoxyribonuclease from cultured cells infected with herpes simplex virus. Biochim. Biophys. Acta, 72:263-276.

is that the virus-induced DNA polymerase does not 'Om- Kit, S., Leung, W.-C., and Trkula, D. (1973) Distinctive properties of plex with other DNA precursor pathway enzymes and mitochondria1 thymidine(dT) kinase from bromodeoxyuridine (dBUF in this respect is distinctly different from the situation resistance mouse lines. Biochem. Biophys. Res. Commun., 54~455-

No evidence indicative Of substrate Of ri- activity copwifies with the DNA polymerase induced by herpes sim-

OuS1y been reported by Others et 1978; Reddy Chiu, C.-S., Cook, K.S., and Greenberg, G.R. (1982) Characteristics of

RNA* This was rendered acid soluble Engstrom, Y., Eriksson, S., Thelander, L., and Akerman, M. (1979)

31:101-113.

in the uni&ected cell.

ACKNOWLEDGMENTS We are indebted to the Science and Engineering Re-

search Council for support. We thank Professor H.M. Keir for provision of facilities, Mrs. Mary Evans for assistance with cell culture, and Mrs. Fiona Mitchell for typing the manuscript. We are also indebted to Mr. David Stevenson for his assistance and critical advice during the preparation of the manuscript.

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