the of blomlcu. chemistry vol. 257, no. 24, issue of ... · the journal of blomlcu.chemistry vol....

THE JOURNAL OF Blomlcu. CHEMISTRY Vol. 257, No. 24, Issue of Decemher 25, pp. 15087-15097, 1982 Printed in U.S.A.

Characteristics of a Bacteriophage T4-induced Complex Synthesizing Deoxyribonucleotides*

(Received for publication, March 8,1982)

Che-Shen Chius, Kathleen S . Cook& and G. Robert Greenberg1 From the Department of Biological Chemistty, The University of Michigan, Ann Arbor, Michigan 48109

A preparation of bacteriophage T4-induced deoxyribonucleotide synthetase complex is described. This very large complex of enzymes can be separated by centrifugation at 100,000 X g, by sucrose step gradient centrifugation, or with molecular exclusion columns. By direct assay and by unidimensional and two-dimensional acrylamide electrophoretic separations the following T4-coded enzymes were shown to be associated with the complex: ribonucleoside diphosphate reductase, dCMP deaminase, dCTP/dUTPase, dCMP hydroxymethylase, dTMP synthetase, and DNA polymerase. Other phage-coded prereplicative proteins related to DNA replication and other phage functions such as the proteins coded by genes 32,46, rZZA, and rZZB as well as many unidentified proteins were also consistently associated with the isolated fractions. T4 DNA topoisomerase, a membrane-bound enzyme, was found in quantity in all purified fractions of the complex, even in preparations apparently free of membrane and of T4 DNA.

The functional integrity of a segment of the complex was followed by measuring the conversion of [tL3H] CDP to the level of 5-hydroxymethyl dCMP. This series of reactions requires the actions of T4-coded ribonucleoside diphosphate reductase and its associated re- ducing system, dCTP/dUTPase and dCMP hydroxymethylase, 3H being lost to water at the last step. In this reaction sequence an intermediate, [5-3H]dCMP, is maintained at low steady state concentrations, and argument is presented that the synthesis of deoxyribonucleotides is channeled and normally tightly coupled to DNA replication.

One of the primary characteristics of this complex is its ready dissociation on dilution into smaller complexes of proteins and to the free forms of the proteins. That the complex is held together by weak electrostatic forces was supported by its sensitivity to dissociation at moderate salt concentrations. Not only the enzymes required in deoxyribonucleotide synthesis but T4 DNA polymerase, T4 DNA topoisomerase, and a number of other proteins dissociate to varying degrees from the larger complexes under these conditions.

8133, GM25793, and GM29025 from the National Institutes of Health * This work was supported in part at various times by Grants AI-

and Grant PCM77-20291 from the National Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Present address, Molecular Biology Unit, The Upjohn Company, Kalamazoo, Michigan 49001. 5 Recipient of the Burton Baker Fellowship which is supported in

part by Institutional Research Grant IN-40U to The University of Michigan from the American Cancer Society. 1 To whom correspondence may be addressed.

Over the past several years this laboratory has presented a series of experiments and arguments proposing that in phage T4 infection deoxyribonucleotide synthesis occurs in a complex of enzymes catalyzing the reductive and channeled conversion of ribonucleotides to deoxyribonucleotides, which in turn are transferred to the integrated replication complex (1- 7). Our goal has been to define this complex by its properties in in vivo and in situ systems in order to approach the isolation and characterization of the complete system.

Kinetic studies have shown that deoxyribonucleotide synthesis is the limiting factor in T4 DNA replication (2, 4, 8) and have suggested that the period of assembly of the deoxyribonucleotide synthetase complex is described by an initial exponential increase in the rate of synthesis of deoxyribonucleotides (2, 8). Phage-induced synthesis of deoxyribonucleotides is controlled at a constant ratio of dTMP:HmdCMP’ derivatives of 2.1:l corresponding exactly to the ratio in T4 DNA, even on infection by Dna- phage (5). Other experiments from this laboratory and that of C. K. Mathews have indicated obligatory interactions between dCMP hydroxymethylase and the replication system (1,9) and the remainder of the deoxyribonucleotide synthetase complex itself (2, 10). Possible evidence for an interaction between T4-coded DNA polymerase and the deoxyribonucleotide synthetase complex has been reported (4) as well as an interaction between dCMP deaminase and another part of the complex (7). Chao et al. have provided genetic grounds for a close association between dCMP hydroxymethylase and T4 DNA polymerase (11). Re- cent in vivo studies from this laboratory represent strong argument that DNA functions in the deoxyribonucleotide synthetase complex, perhaps by action through ribonucleoside diphosphate reductase (6).

An in vivo investigation by Chiu et al. of infection by phage T4 nrd mutants has demonstrated that deoxyribonucleotide synthesis is reduced to about 25% of the wild type value, the remainder representing the activity of host ribonucleoside diphosphate reductase (12). The experiments have shown that T4 ribonucleoside diphosphate reductase is the limiting activity in the system synthesizing deoxyribonucleotides.

Since 1976 our laboratory has been engaged in the isolation of the T4 deoxyribonucleotide synthetase complex and in a study of several of its protein components. Preliminary reports of the isolation studies have been made (13, 14). From our first report T4 DNA polymerase has been associated with the preparations. In 1978 we reported briefly that other phage T4 gene products implicated in DNA replication were also found in the fraction (14). We wanted to assure ourselves that the preparations were not artifactual and that the enzymes were not carried by an aggregate of host components as a vector.

The abbreviations used are: HmdCMP, 5-hydroxymethyl dCMP; Dna-, no DNA synthesis phenotype; pnrdA, the protein coded by gene nrdA; ptd (dTMP synthetase), protein of gene td; SDS, sodium dodecyl sulfate.

15087

15088 Phage T4 Deoxyribonucleotide Synthetase Complex

During the period of these studies new insight into the func- tion of DNA (6) and other regulatory factors (5, 7) in the complex and the possible role of the phage-induced DNA topoisomerase (15, 16) in deoxyribonucleotide synthesis (8) increased the ultimate intricacy of the complex. Mathews and coworkers in the intervening time have published accounts of aggregates of the deoxyribonucleotide-synthesizing enzymes in which some coupled reactions were described (10, 17) and in which T4 ribonucleoside diphosphate reductase was reported to play a part (18).

The present paper describes the isolation from phage T4- infected cells of an aggregate of enzymes in which we measure the reductive conversion of CDP to HmdCMP. This preparation appears to be free of T4 DNA and cytoplasmic membrane. T 4 D N A polymerase and many of the components of the replication complex have been identified in the preparation. These experiments in conjunction with earlier findings suggest that the conversion of CDP to the level of HmdCMP is via a channeled process. A salient feature of the deoxyribonucleotide synthetase complex is its sensitivity to dissociation by dilution and by relatively low concentrations of elec- trolytes to yield free enzymes and intermediately sized complexes. T4-coded ribonucleoside diphosphate reductase appears to be an integral part of the active complex and to be easily dissociated from it.

EXPERIMENTAL PROCEDURES

The bacterium employed in all experiments was Escherichia coli thyA deoB (strain GM 201), derived from strain B and described earlier (19,20). Bacteriophage T4D and the amber mutants were used earlier (2). amN82 is a mutant of the dna gene 44, amElO, dna gene 45, and am4335, gene 43 (DNA polymerase). Amber mutants were grown on the supD suppressor strain, E . coli CR63. Growth of phage and the conditions of infection have been described (2, 12). The multiplicity of infection was 8, the temperature was 30 “C, and the period of infection was 30 min.

Crude extracts were prepared by grinding the pellets from infected cultures (3 X 10” cells) with about 2 volumes of bacterial grade aluminum oxide powder at 4 “C. The paste was extracted with 3 ml of 50 mM Tris-C1, pH 7.8, and 8 mM dithiothreitol. The aluminum oxide and cell debris were removed by centrifugation at 10,000 X g for 10 min.

A preparation referred to as the 100,000 X g pellet was obtained by treatment of infected cells with EDTA, egg white lysozyme, and Triton X-100. The cell pellet from 300 ml of infected cells (5 X IO8 cells/ml) was suspended in a final volume of 3 ml of 40% sucrose (w/ w, n = 1.3998 at 20 “C) and 10 mM Tris-CI, pH 7.4. A lysozyme solution of 1 mg/ml in 20 mM EDTA was added to give 150 pg/ml of lysozyme and 3 mM EDTA. After incubation at 30 “C for 15 min, a solution of 40 mM KC1 and 10 mM Tris-C1, pH 7.4, was added to a fmal volume of 5 ml. A solution of 20% Triton X-100 (w/v) was added to a concentration of 1%, and the mixture was allowed to stand at 4 “C for at least 15 min. In earlier experiments 0.7% Brij-58 was employed. After centrifugation 3 times at 15,000 X g for 10 min, the supernatant was centrifuged at 100,OOO X g for 2 to 3 h. The 100,000 x g pellet was dissolved in 0.5 ml of 3% sucrose containing 20 mM KC1, 10 mM Tris-C1, pH 7.4, and 1 mM EDTA. In all experiments the EDTA stock solutions were adjusted to pH 7.4.

Thymidylate synthetase and dCMP hydroxymethylase were assayed by following the release of tritium from [5-3H]dUMP and [5- 3H]dCMP, respectively, as described earlier (2). Because of variations in the activity measurements of dTMP synthetase from run to run, the activities of this enzyme were normalized to.be the same as those of dCMP hydroxymethylase in extracts. The absolute values for dTMP synthetase may vary by a factor of 2. T4 DNA polymerase was assayed according to Goulian et al. (21). Because of the importance of the nature of the primer-template in the rates attained, the values presented compare only the relative activities of different fractions. dCMP deaminase was measured by a modification of the procedure of Scocca et al. (22), using [5-3H]dCMP as the substrate and separating the product by thin layer chromatography in a solvent of l-butanol:methanol:H20:conc HC1, 701015:5, v/v. Dihydrofolate reductase was measured according to Warner and Lewis (23). Deox- ycytidine deaminase was assayed spectrophotometrically (24). Deox-

ycytidine triphosphatase was measured as described previously (2). The presence of cytoplasmic membrane in various fractions was monitored by following the marker enzyme, NADH oxidase, according to the procedure of Osborn et al. (25). The conversion of CDP to HmdCMP was measured by a modifcation of the procedure of Yeh et al. (26), using [5-3H]CDP as substrate. The reaction mixture contained 0.1 mM dATP, 10 mM MgC12,8.5 mM Tris-C1, pH 8.5, 1 mM NADPH, 0.7 mM EDTA, 0.4 mM [5-3H]CDP, 10-20 cpm/pmol, and preparations of phage T4 thioredoxin (27) and host thioredoxin reductase and the deoxyribonucleotide synthetase fraction in a reaction volume of 0.10 to 0.12 ml and at a temperature of 30°C.

E. coli thioredoxin reductase was purified by the procedure of Moore et al. through the DEAE-cellulose chromatography step (28). An aliquot of this fraction added in each ribonucleotide reductase assay had an activity effecting a change of 0.1 absorbance unit per min at 412 nm in the 5’,5-dithiobis(2-nitrobenzoic acid) measurement of thioredoxin reductase. In these studies T4 thioredoxin (27) normally was a crude preparation obtained from E. coli B cells infected by phage SP62 amN55 (regA 42-) a t 37 “C for 30 min. A sonicate of 1.5 X 10” cells per 4 ml of 0.05 M potassium phosphate buffer, pH 7.6, was prepared, centrifuged at 5,000 X g for 5 min, heated at 65 “C for 5 min, and centrifuged at 10,000 X g for 10 min. The ribonucleotide reductase assay employed an aliquot of the resulting supernatant, usually 10 pl, causing a change in absorbance of 0.015 per min at 412 nm in the presence of excess thioredoxin reductase in the standard assay. In this system the same results have been obtained with highly purified preparations of T4 thioredoxin and thioredoxin reductase. The CDP to HmdCMP reaction was terminated by the addition of 30 pl of 50% trichloroacetic acid. 3H release was followed by the charcoal adsorption method (2), and dCMP was separated from the substrate by thin layer chromatography on Eastman Kodak plastic sheets precoated with cellulose in a solvent composed of 30 volumes of 1 M ammonium acetate a t pH 9 with 10 mM EDTA, saturated with sodium tetraborate, plus 70 volumes of 90% ethanol. [5-3H]dCDP was not detectable in these reaction mixtures. The product of the CDP + dCDP reaction is coupled to dCTPase and dCMP hydroxymethylase, both having greater activities than the fmt step in these fractions. Thus the reaction sequence is [5-3H]CDP ”* [5-3H]dCDP + [5-3H] dCMP --f [HmdCMP] + 3H (as 3HOH), and ribonucleoside diphosphate reductase activity can then be accounted for by the measurement of 3H released into water plus the small quantity of [5-3H]dCMP found. The significance of the measurement of 3H released to the rate of formation of HmdCMP, the use of the designation [HmdCMP], and the reason that an added tetrahydrofolate system is not required, are treated under “Results.”

Linear sucrose gradients consisted of 5 ml of 7 to 25% (n = 1.3433 to 1.3723) sucrose. 0.3 ml of the 100,000 X g pellet fraction was layered onto the gradient and centrifuged in a Beckman-Spinco rotor SW 50.1 at 100,000 X g for 12-14 h at 4 “C. Sucrose step gradients were carried out with a 0.5-ml60% sucrose cushion, 2.0-ml20% sucrose, and a 3.0- ml sample of the supernatant of the 15,000 X g centrifugation diluted to contain 15% sucrose, centrifuging in the SW 50.1 at 150,000 x g for 15 h. Sucrose solutions contained 20 mM KC1, 10 mM Tris-C1, pH 7.4, and 1 mM EDTA. Bovine liver catalase (Mr = 240,000) was used as a molecular weight marker in sucrose gradient centrifugations and in

spectrophotometrically. A sample of purified T4 dCMP hydroxy- molecular exclusion column separations, and its activity was measured

methylase was a kind gift from Michael Bittner and Bruce Alberts, Department of Biochemistry and Biophysics, University of California, San Francisco.

Early phage proteins were labeled by addition of 35S04-2 to the culture (final concentration, 10-20 pCi/ml) 2 min after infection with an amber Dna- mutant, and the infection was continued to 30 min. In many instances 30 to 60 ml of infected cells were exposed to 100 to 200 pCi/ml, and at the end of the incubation period the separated labeled cells were mixed with the centrifuged cells of a 10 times larger culture of unlabeled infected cells. The minimal salts medium of Vogel and Bonner (29) was used with MgC12 substituted for MgS04 and supplemented with 3 X M Na2SOI. Electrophoresis was carried out with 10% acrylamide slab gels containing 0.1% SDS in the buffer system of Laemmli (30). Two-dimensional electrophoresis was carried out by a modification (31) of the nonequilibrium pH gradient procedure of O’Farrell et al. (32). Kodak RP/R-54 x-ray f h was used for autoradiography.

RESULTS

Conversion of CDP to [HmdCMPj by 100,000 X g Pellet Fraction-In earlier experiments we had found that in the

Phage T4 Deoxyribonucleotide Synthetase Complex 15089

measurement of the sequence, [5-3H]CDP + [5-3H]dCDP +

[5-3H]dCMP e [HmdCMP] (see below) in crude extracts, dCMP is maintained at very low levels during the linear synthesis of the product (14). In Fig. 1 the kinetics is examined in a 100,oOO X g pellet obtained from cells infected by amN82, a gene 44 (Dna-) mutant (see under “Experimental Proce- dures”). The steady state concentration of dCMP was 7 to 9 PM while the synthesis of HmdCMP with this amount of preparation reached about 3 nmol per min per ml with a lag of only about 1 min before reaching the constant rate. This rate, which remained constant for 20-25 min, corresponds to about 275 molecules of product/cell/s and is a measure of the activity of T4 ribonucleoside diphosphate reductase, the rate- limiting enzyme. Other measurements have shown an apparent K , value of about 45 PM for dCMP in the dCMP hydroxymethylase reaction in crude extracts (not presented). Inter- fering reactions were unlikely in this K,,, measurement, and the data corresponded with earlier unpublished results with the purified enzyme (33). Thus the steady state concentration of dCMP was far below the apparent K,,, value.

In these assays the activity of host ribonucleoside diphosphate reductase was eliminated by using dATP which acti- vates the phage enzyme but completely inhibits the host enzyme (34, 35). That the phage-coded enzyme was being measured and that the host enzyme was inhibited by dATP was verified in complementation studies using crude extracts of cells infected by phage nrdB and nrdA2 mutants (12).

These fractions contain sufficient quantities of tetrahydrofolate derivative for exchange of 3H of [5-3H]dCMP with water by dCMP hydroxymethylase (33). However, when the substrate was 14C-CDP and I4C-HmdCMP formation was measured, tetrahydrofolate and serine as a 1-carbon donor were necessary further additions (not presented). Yeh and Green- berg mentioned such requirements in earlier studies on dCMP hydroxymethylase (33).

The pellet fraction, in addition, carries out the conversion of UDP to dTMP (see also Ref. 18). This reaction sequence depends not only on added T4 thioredoxin, host thioredoxin reductase, and NADPH, but also on tetrahydrofolate and serine (not shown). A substrate level requirement for tetrahydrofolate is expected since dTMP synthetase converts tetrahydrofolate (as the 5,lO-methylene derivative) to dihydrofolate, and dihydrofolate reductase is not present, as described below. In experiments in which serine was added, rabbit liver serine hydroxymethylase, generously provided by Vern Schirch, Virginia Commonwealth University, Richmond (36), was also added, though it was not established that the corresponding E. coli enzyme was absent from the complex.

Since dCMP hydroxymethylase is present in the pellet, the dCMP pool formed by the reductive pathway can be siphoned off. However, in the absence of added 1-carbon donor, dCMP merely exchanges its 5-hydrogen atom with Hz0 in direct proportion to dCMP hydroxymethylase activity without oth- erwise being altered (33). Thus the reaction sequence being studied is [5-3H]CDP + [5-3H]dCDP 4 [5-3H]dCMP - [5-’H]dCMP + 3HOH. In the dCMP hydroxymeth-

+‘HOH

The phage T4 genes referred to in this paper and their protein products or their functions are: gene 43, DNA polymerase; gene 44, protein required in DNA replication; gene 45, protein required in DNA replication and late transcription factor; gene 32, single strand binding protein; rIIA, a membrane protein, rapid lysis, rIIB, a membrane protein, rapid lysis; gene 46, arrest of DNA synthesis, host DNA degradation; gene 30, DNA ligase; genes 39, 52, and 60, protein subunits of DNA topoisomerase; gene 41, DNA replication initiation factor; gene 42, dCMP hydroxymethylase; nrdA and nrdB, protein subunits a and /3, respectively, of ribonucleoside diphosphate reductase; ipIZZ, unprocessed T4 internal protein.

- X ” - _

8 I

m - - I

I I I I

IO 20 3 0 MINUTES

FIG. 1. Reductive conversion of CDP to the level of HmdCMP by the 100,000 X g pellet fraction from phage T4 Dna--infected cells. This procedure and the preparation of the 100,000 X g pellet obtained from the 15,000 X g supernatant after EDTA, lysozyme, and Triton X-100 treatment are described under “Experimental Proce- dures.” The phage was the Dna- mutant, amN82 (gene 44). The reaction mixture per ml contained the pellet fraction isolated from 1.1 X 10” cells (but see under “Enzymes of the 100,000 X g Pellet”).

ylase step HmdCMP is not formed, but the rate of exchange of 3H with HOH is an accurate measure of the activity of the enzyme in the pathway. Inhibition by HmdCMP cannot occur (see under “Experimental Procedures” and Ref. 33), nor does the dCMP-’H product affect the rate of utilization of the dCMP-3H intermediate. Therefore, in describing the assay of this sequence of reactions [HmdCMP] is bracketed. In this coupled sequence the rate of 3H release from [5-3H]dCMP is dependent on the rate of ribonucleoside diphosphate reductase. The second reaction is very fast, and [5-3H]dCDP is ordinarily not detected. [EJ-~H]~UMP is not formed from 15- 3H]dCMP by escape through dCMP deaminase since the latter enzyme requires HmdCTP for activation (7). In confii- mation of this statement, [5-3H]dUMP is not detected even though it cannot be converted to dTMP.

Enzymes of the IO0,OOO X g Pellet-Repeated analyses of the 100,OOO x g pellet and its supematant have shown that the enzymes of the deoxyribonucleotide synthesis pathway are consistently enriched in the pellet. The 15,000 X g supernatant from cells infected by amElO (gene 45, Dna-) was centrifuged at 100,000 X g for 3 h. In these experiments the percentages of several phage-coded enzymes occurring in the pellet relative to the supernatant ranged as follows: dCMP hydroxymethylase, 9-25%; dTMP synthetase, 23-35%, dCMP deaminase, 21-35%, and ribonucleoside diphosphate reductase, 11-17%. In the same experiments only 1 to 2% of the phage-coded dihydrofolate reductase activity and 0 to 1% of deoxycytidine deaminase, a host enzyme, were found in the pellet.

It is important to note that under the conditions of centrifugation, only 3 hours at 100,000 X g in the presence of 22% sucrose, the complexed proteins are brought down relatively slowly and incompletely to form a pellet. (See legend to Table I for effect of 12-h centrifugation.) In the section below in which sucrose linear and step gradients are employed it is clear that greater centrifugal force and longer times of centrifugation result in a far greater yield of the complexed fractions.

Using amN82 for infection, the 100,000 x g pellet and its supernatant were examined by two-dimensional electrophoresis. The expected contamination of a given volume of pellet


FIG. 2. Autoradiogram of two-dimensional electrophoretic separation of the S6S-labeled prereplicative proteins of the 100,000 X g pellet after infection by amN82. Scored known T4 gene products and unidentified protein chains (indicated by unmarked arrows) are enriched in the pellet. Unmarked spots represent contamination from the supernatant (see text). The asterisks indicate proteins found exclusively in the pellet. The position of the protein product of gene 44 is shown by a circle. Gene 41 protein is normally found as a doublet (31).

by the supernatant fraction (electrophoresis not shown) was taken into account on redissolving the pellet for analysis. We tentatively consider the protein products of the following T4 genes to be enriched in the 100,OOO X g fraction by this form of analysis (Fig. 2) and by enzymatic assays: 43, 30, 32,39,52, 60, 41, 42, 46, nrdA, nrdB, rZZA, rZZB, and ipZZZ. In addition we have designated 19 other unidentified proteins that appear to be enriched in the pellet. The unmarked spots are considered to represent contaminating protein chains from the supernatant. A number of proteins occurred only in the supernatant, thus representing further support for the conclusion that the proteins found in the pellet are not merely occluded or contaminants from the supernatant. At the same time a few proteins were found almost exclusively in the pellet (e.g: prIIB and those marked with asterisks). In these preparations pl, the T4-coded deoxyribonucleotide kinase, was found to be very low. This enzyme clearly was present in the fractions described by Reddy and Mathews (10) and Allen et al. (18). Including dCMP deaminase, dCTP/dUTPase, and dTMP synthetase, we account for some 35 phage-coded protein chains in the 100,OOO X g pellets, approximately half of the prereplicative spots resolved by this procedure (31). This is an unusually large number of protein chains. Some are expected to be components of multisubunit enzymes (DNA topoisomerase, ribonucleoside diphosphate reductase). I t should be emphasized that these findings do not constitute argument that all of the members of this large group of phage- coded proteins are associated with the deoxyribonucleotide synthetase complex. However, they support the idea that the synthetase complex and the replication enzymes are associated and cosediment in large aggregates. As an adjunct, the results bear out the suggestion by Hibner and Alberts that the T4 replication enzymes exist as a complex (37).

In another study removal of T4 ribonucleoside diphosphate reductase from the 100,OOO X g pellet by passing the preparation through a dATP agarose column, which adsorbs the enzyme by binding its a2 subunits, did not disrupt the structure of the aggregate of proteins. Except for the reductase, the same protein chains observable by one-dimensional SDS electrophoresis were present in the pellet when reisolated by centrifugation at 100,OOO X g in 5% sucrose for 2.5 h (38).3 A

K. S. Cook and G. R. Greenberg, manuscript in preparation.

comparable result was obtained with amB55, an amber mutant of nrdB (coding for the /I2 subunits), in that the constituents of the isolated pellet were unchanged even though the pellet lacked both the a2 and /I2 subunits of the reductase.“ This appears to differ from a report in a recent paper that infection by amB55 prevented the isolation of the deoxyribonucleotide-synthesizing enzymes as an aggregate (18) and may represent a difference in the conditions of isolation of the preparations (see under “Discussion”).

Separation of Deoxyribonucleotide-synthesizing Complex by Sucrose Gradient Centrifugation-If the 100,OOO X g pellet is taken up in a sucrose solution containing 10 mM Tris-C1, pH 7.4, 20 mM KC1, and 1 mM EDTA and subjected to separation by centrifugation in a 7 to 25% sucrose gradient, a large number of proteins separate as heavy fractions with some proteins appearing as complexes with lower sedimenta- tion values or as their free forms. Fig. 3 shows the distributions of activities of dCMP hydroxymethylase, dTMP synthetase, and T4 DNA polymerase on sucrose gradient separation in the 100,OOO X g pellet isolated from cells infected by amElO. Under these conditions of centrifugation the enzyme activities fell in several peaks. From repeated separations of this type, dTMP synthetase and DNA polymerase were in the heaviest fraction with some overlap of dCMP hydroxymethylase. The three enzymes comigrated as a part of an intermediate peak(s) in fractions 4 to 8. In the region between fractions 12 to 17 these enzymes occur as their free forms. Little or no dTMP synthetase was present as the free enzyme. Some of the activities are spread between the region of free enzyme and the intermediate peak, perhaps as smaller complexes. An analysis by SDS-acrylamide electrophoresis of the sucrose gradient fractions from a comparable separation after amN82 infection presented in Fig. 4 shows that the proteins coded by genes 43, nrdA, 39, 52, 46, td, ipIII, and 42 (39), which were resolvable, were found in the intermediate fraction and that most of these as well as many unidentified proteins were also in the heaviest fraction. However, nrdA protein was absent from the heaviest fraction and was present in lesser amounts in the intermediate fraction than other deoxyribonucleotide- synthesizing enzymes, e.g. p42 and ptd, but appeared to occur mainly in its free a2 form in fractions 11 to 15.4 A number of

‘T4 a2 does not dissociate into its subunits readily (35). The purified enzyme (ad2) sediments to the position of catalase?

300

v) W

!z 200 - > + 0

- a W 5 >- N

100

I

Phage T4 Deoxyribonucleotide Synthetase Complex

I I I

dCMP Hydroxymethylose

o d T M P Synthetose

A T4 DNA Polymerase

15091

N

5 IO 15 2 0

FRACTION NUMBER FIG. 3. Distribution of dCMP hydroxymethylase, T4 dTMP arbitrary and relative and show only distribution. These activities are

synthetase, and T4 DNA polymerase activities in a linear on the same scale as the results shown in Fig. 7 which were obtained sucrose gradient centrifugation of the 100,000 X g pellet from simultaneously with the same preparations. Interposed on this graph cells infected by amEl0 ( d m gene 45). The sucrose gradient is a separate run in which an amber mutant of gene 43 (DNA contained 20 n" KC1, 10 m~ Tris-C1, pH 7.4, and 1 mM EDTA. The polymerase) was used to demonstrate that host polymerases do not preparation of the sucrose gradient is described under "Experimental significantly interfere with the assay. In a separate run of 100,OOO X Procedures," and centrifugation was for 12.5 h. Enzyme activities are g for 19 h, purified dCMP hydroxymethylase peaked at tube 13.

FIG. 4. SDS acrylamide gel electrophoresis of the S5S-labeled proteins from fractions obtained by separation of the 100.000 X g pellet on a linear sucrose gradient. Infection was with amN82. Catalase peaked at fraction 11. Recently this laboratory reported that in crude extracts the second protein band after p43 is the product of nrdA (12).3 In other electrophoretic separations of comparable sucrose gradient runs the protein product of rUA was readily resolved (between bands 2 and 3; see Fig. 2 and Ref. 39) and was found mainly in the heavy fractions of the gradients.

1 = 43 2= nrdA

4= 46 6= 39 7 = 52

"""4 ""

Whble 100 ppt 1 3 5 7 9 1 1 13 15 17 Whole Prep kg Prep

I t

Pellet FRACTION NUMBER

experiments indicated that dilution of the complex of enzymes To obtain more concentrated preparations so as to increase leads to partial dissociation of the complex to lighter fractions. the association of the components of the complex and yet to Apparently one of the f i t to dissociate or the last to associate remove the complex more effectively from other smaller frac- is T4-coded ribonucleoside diphosphate reductase (Fig. 4). tions, the 15,000 X ,q supernatant was adiusted to 15% sucrose


and layered onto 20% sucrose over a 60% cushion in a centri- fuge tube. This step gradient was subjected to centrifugation at 150,000 X g for 15 h. By this procedure a rather large portion of the complex can be concentrated at the cushion. Fig. 5 shows the distribution of phage T4-coded dTMP synthetase, dCMP hydroxymethylase, the CDP -+ [HmdCMP] reaction, dCTPase, and T4 DNA polymerase activities. dCMP hydroxymethylase is either present in considerable excess or is readily dissociated from the complex. The maximum activities of the several enzymes, except dCTPase, correspond to the peak of the activity of the sequence CDP -+ dCDP -+

dCMP + [HmdCMP]. The indicated peak rates of this sequence of reactions are equivalent to about 780 molecules of product per s per original infected cell. While dCTPase fell primarily in fractions 2 to 9, it showed a shoulder in fractions 10 and 11. T4 ribonucleoside diphosphate reductase, though peaking in tubes 10 and 11, was the limiting step in the sequence, since dCTPase and dCMP hydroxymethylase activities were clearly greater than that of the reductase in the complex and since intermediates did not accumulate.

Table I summarizes the activities of dCMP hydroxymethylase and T4 ribonucleoside diphosphate reductase (measured as the CDP + [HmdCMP] reaction) in the various fractions described in this section, expressed in nanomoles of product per min per 10" infected cells. T4 ribonucleoside diphosphate reductase is especially labile at the stages of the 15,000 x g, 100,OOO X g, and sucrose gradient centrifugations. Accordingly the activities vary widely. After purification through a dATP- Sepharose affinity column this enzyme is stabilized considerably (34) ?

Separation on Agarose Columns-When the aggregate of proteins in the pellet fraction was passed through an A-1.5m agarose gel permeation column (Bio-Rad Laboratories, Rich- mond, CA), dCMP hydroxymethylase, dTMP synthetase, and T4 DNA polymerase were excluded in the void volume in

keeping with a particle of greater than 1.5 X lo6 daltons (Fig. 6, bottom). Smaller portions of the enzyme activities were found in fractions corresponding to their free forms as well as in complexes in the intermediate region. In Fig. 6, the top

TABLE I Representative distributions of ribonucleoside diphosphate

reductase and dCMP hydroxymethylase in fractions from infected cells after lysis b r lvsozvme-EDTA-Triton X-100 treatment

nmol per min per 10" cn- fected cells

Lysed extract

100,OOO X g centrifugation of 15,000 X g 15,000 X g supernatant 670" 39- 170

supernatant Supernatant (3 h) 690,' 615 20'-75 Pellet (3 h)' 67'-165 2.7b-23

Fractions above cushion 290" 4.9" Fractions at cushion 266" 8.7"

15,000 X g supernatant on step gradient

a These values represent one experiment (see Fig. 5). dCMP hydroxymethylase in the 15,000 X g pellet in various experiments represented IO-40% of the activity. dCTPase activity in the 15,000 X g supernatant was calculated to be 6400 nmol/min/lO" infected cells. Ribonucleoside diphosphate reductase activities indicated by are based on the CDP + [HmdCMP] reaction; the other values represent the sum of labeled dCMP and 3H released. The amount of dCMP was usually very low.

* These values represent one experiment. In another study, when the time of centrifugation at 100,000 X g

was increased to 12 h, the pellet contained 67% of the reductase activity, 81% of the T4 DNA polymerase activity, 91% of the dTMP synthetase, and 47% of the dCMP hydroxymethylase (see text). In an extract prepared by sonication the total activity of T4 ribonucleotide reductase was 112 nmol/min/lO" infected cells.

6 2 FRACTION NUMBER

FIG. 5. Distribution of dCMP hydroxymethylase (Hmase), dTMP synthetase, dCTPase, T4 DNA polymerase and the reaction sequence, CDP + dCDP + dCMP + [HmdCMP], in a sucro~e step gradient centrifugation. Samples were taken in order from the top of the gradient. The significance of the designation [HmdCMP] is described in the text. The reaction time for measurement of the CDP ++ [HmdCMP] sequence was 10 min. The sample was the 15,000 X g supernatant fraction corresponding to 180 ml of

culture infected with phage amN82 and treated with EDTA, lysozyme, and Triton X-100 as described. The values for the CDP + [HmdCMP] sequence are per min reaction time per total sucrose gradient sample. dCMP hydroxymethylase, measured at saturation concentrations of labeled dCMP, is expressed in nanoatorns of 3H released per min per sucrose gradient sample and dTMP synthetase is in the same units. dCTPase and DNA polymerase are expressed in units per min X the indicated graphical factors.


200-

dCMP Hmase o dTMP Synthetase A T4 DNA Polymerase

‘ O ( I

FRACTION NUMBER FIG. 6. Chromatography of 100,000 X g pellet fraction on a

Bio-Gel A-1.Sm column. 0.25 ml of 100,OOO X g pellet fraction from cells infected with amN82 was applied to a Bio-Gel A-1.5m column, 0.7 X 18 cm. Elution was with 5% sucrose, 20 mM KC1,lO m~ Tris-C1, pH 7.4, and 1 mM EDTA at 0.15 ml per 12 min, and each fraction contained about 0.15 ml. Enzyme activities are expressed as in the legend to Fig. 3. Catalase was the marker; a small part fell as a separate peak in the void volume. From control runs, the peak of the void volume was at fractions 12 and 13 (dextran blue), and bovine serum albumin would peak at fraction 21. Hmase, hydroxymethylase.

frame shows that the complex catalyzing the reaction sequence CDP + dCDP + dCMP + [HmdCMP] falls primarily in the void fraction. The lack of clear separation of the large fraction from the free enzymes is consistent with its partial dissociation to smaller complexes (see Fig. 3). Control studies showed that the column easily separated blue dextran from serum albumin. However, the observed specific activity of the pathway in the excluded fraction, considering the loss during centrifugation of the 100,OOO X g pellet, did not differ greatly from that observed in sucrose step gradients. Taken together fraction numbers 10 through 14 give a value of about 270 molecules of product per s per original infected cell.

Effect of Ionic Strength on the Stabilities of Complex-In respect to many of its enzymes the complex fractions are exceptionally sensitive to ionic strength. Both lower and higher levels of salt than those employed in the experiments described previously cause dissociation into smaller forms or to the free forms. In 5 m~ Tris-C1, pH 7.4, the dCMP hydroxymethylase, dTMP synthetase, and T4 DNA polymerase activities of the 100,000 X g pellet were found to be approximately one-third the levels found with 10 mM Tris-C1 buffer plus 20 mM KC1. Later experiments have indicated that the optimum KC1 concentration in the buffer solution to maintain the complex may lie between 40 and 60 m~ rather than 20 mM. Increased dissociation begins at some concentration between 60 and 90 mM. Fig. 7 shows the effect of 0.12 M KC1 in 10 m~ Tris-C1 and 1 mM EDTA. The three enzymes are

displaced from the most rapidly sedimenting fractions and appear in a considerably more slowly sedimenting fraction(s). Autoradiograms of the electrophoretic separations of the fractions of comparable linear sucrose gradients showed that many proteins have moved completely or partially from the heavy fractions to intermediately sized fractions and to their free forms (not presented). These included p39, p52 (15, 16); p43, pnrdA, ptd, p42, and unidentified proteins. prIIA, pipIII, p46, and a number of unidentified proteins were not displaced or moved only slightly.

On raising the KC1 concentration to 0.2 M or on using 0.1 M MgC12 all of the analyzed enzymes of the larger and of the intermediately-sized complexes appeared as their free forms (Fig. 7, inset).6 Analysis of the distribution of 35S-labeled proteins formed after phage infection in a linear sucrose gradient separation carried out in the presence of 0.25 M KC1, 0.04 M N,N-bis(2-hydroxyethyl)glycine buffer, pH 8.5, and 1 m~ EDTA demonstrated that 77% of radioactivity had moved to the fractions corresponding to the free forms of the proteins (fractions 13-20; catalase peak at fraction 10). One-dimensional electrophoresis of the gradient fractions and autoradiography showed that the preponderance of the proteins in the heavy and intermediate fractions, including prIIA, p46, pipIII, p39, p52, and unidentified bands, had dissociated to the free forms of the proteins or enzymes (not shown). Com- pared to 0.02 M, KC1 at 0.2 M inhibited T4 ribonucleoside diphosphate reductase in a 100,000 X g pellet fraction by 77% and dCMP hydroxymethylase by 61%. 0.1 M MgC12 inhibited the reductase almost quantitatively, in confirmation of the report of Berglund (35).

Distribution of Phage DNA in Sucrose Gradients; Possible Contaminants of the Complex Fractions-In order to follow the distribution of T4 DNA during isolation of the complex, the infection was carried out with DNA-labeled phage. The labeled phage were prepared by infecting E. coli CR63 with phage amElO in the presence of 20 pCi of r3H]thymidine per ml of culture and purifying the resulting phage by differential centrifugation. Infection by labeled amElO and analysis of the 100,000 X g pellet on a linear sucrose gradient was as in Fig. 3. After lysozyme-EDTA-Triton X-100 treatment approximately 95% of the labeled phage DNA remained in the 15,000 X g precipitate. Of the remaining 5% in the supernatant, 14% (or 0.7% of the total) entered the 100,000 X g pellet. 97% of the DNA of this fraction centrifuged to the bottom of a 7 to 25% sucrose gradient in 14 h at 100,OOO X g under the conditions employed in Fig. 3 (graph not shown). That DNA centrifuges to the bottom is shown in Fig. 4. p32 and pipIII, both of which bind tightly to DNA, are the predominant bands in the pellet fraction of the gradient (marked ppt). In these measurements of the distribution of T4 DNA, the rapidly sedimenting fraction (monitored by its dCMP hydroxymethylase activity) occurred mainly in fractions 3 to 8 from the bottom in a total of 17 tubes, and the catalase marker was at fractions 10 and 11. Less than 1 part in 10,000 of the original T4 DNA was present in the rapidly sedimenting fraction, and the very low background DNA counts showed no peak throughout this fraction. A similar experiment was performed to follow the distribution of T4 DNA after infection by T4D (wild type). In this study newly synthesized DNA was labeled by r3H]thymidine (2 pCi/ml of culture) during infection. Again 97% of the DNA associated with the 100,000 X g pellet sedimented to the bottom of a sucrose gradient. While it is

5A. F. Seasholtz and G. R. Greenberg, unpublished results. In addition, p60, p39, and p52 remain associated through all the manip- ulations of this study.

These enzymes are reassociated by dialysis of the salt and reisolated by centrifugation.


300

FIG. 7. Enzyme activities in fractions from analysis of 100,000 X g pellet in a linear sucrose gradient containing 0.12 M KC1. This experiment was carried out with the Same pel- 1d used in Fig. 3, and all conditions are the same as described in the legends to that figure, except for the additional KCI. In the inset, the sucrose gradient contained buffer with 0.1 M MgC12. The arrows define the peak of the catalase marker. Hmase, hydroxymethylase.

PPt

unlikely that DNA was physically associated with the complex, the actual background DNA counts could still be equivalent to many T4 DNA molecules. However, treatment of the 100,000 X g fraction by pancreatic DNase I had little or no effect on the profie of enzymes formed in sucrose gradients carried out under conditions as in Fig. 3.

Ribosomes represent another possible contaminant. These components have a propensity for binding early proteins synthesized after phage T4 infection (40). However, when ribosomes purified from uninfected cultures were added to extracts from 35S-labeled infected cells and reisolated by differential centrifugation, little or no early protein was adsorbed (not shown). In another set of experiments, host RNAs were prelabeled with [5-3H]uracil during exponential growth followed by exposure of the growing culture to high concentrations of unlabeled uracil for 15 min to dilute out pools of free nucleotides. On separation of the 100,000 x g pellet by sucrose gradient centrifugation the labeled RNA fractions, which of course primarily represented ribosomes, did not correspond with the position of the complex of T4 enzymes (not presented). While ribosomes were present as broad peaks that partially overlapped the complex, the two fractions did not actually coincide, and their relationship to one another was not constant. Thus small changes in KC1 concentration, e.g. 0.02 to 0.06 M (see section on ionic strength), significantly moved the position of the complex without altering the position of the ribosomes, which remained relatively constant, at least between 0.02 and 0.12 M salt (not shown).

It should be emphasized that the deoxyribonucleotide synthetase complex fraction as separated by linear sucrose gradient centrifugation contains a large number of host proteins. This is apparent from the Coomassie blue staining of SDS acrylamide electrophoresis gels and more specifkally from prelabeling of the host proteins and autoradiograms of the electrophoresis gels (not presented). The occurrence of host proteins in these fractions may not have been appreciated in earlier papers (10, 17, 18).

In order to assess the membrane content of the 100,000 X

5 10 15 20 FRACTION NUMBER

g fraction, NADH oxidase, a host integral protein of the cytoplasmic membrane, was measured (25). Samples of the 100,000 X g pellet corresponding to lo9 cells infected by amElO (Dna-) showed no detectable activity, whereas membrane fractions prepared by a modification of the method of Craine and Rupert (41) and corresponding to the same number of infected cells gave activities of 0.4 to 0.6 absorbance units/min at 25 "C.

DISCUSSION

The primary findings in this paper are that in phage T4 infection the enzymes converting ribonucleoside diphosphates to deoxyribonucleotides occur as an isolatable complex in association with several enzymes involved in DNA replication and that the complex is readily dissociable by dilution and by relatively mild concentrations of salt. This aggregate of proteins clearly is very large and is separated readily from lysozyme-EDTA-Triton X-100 lysates by centrifugation at 100,OOO X g. By two-dimensional electrophoresis many prereplicative proteins appear to be enriched in the pellet (Fig. 2). In the heavy fractions of the sucrose gradients (Fig. 3) not only are the phage-coded enzymes converting ribonucleoside diphosphates to deoxyribonucleotides found together, but T4 DNA polymerase, p32, p46, p39, and p52, which have direct or accessory roles in replication, are also found as major proteins. In addition prIIA, pipIII, and as yet unidentified bands are found associated with these fractions (Fig. 4). It is clear that other components of the replication system may be present in these fractions but are not readily separable by unidimensional electrophoresis (see Fig. 2). Phage-induced dihydrofolate reductase was not found in these fractions in agreement with the finding of Reddy et al. (17, 18). These workers also have demonstrated the presence of the host nucleoside diphosphate kinase in their complex fractions.

I n situ studies (3) argue that the pathways from ribonucleoside diphosphates to deoxyribonucleoside triphosphates and thence to DNA are channeled (see below). However, extensive kinetic studies are needed to assess the accessibility


of the intermediates in these pathways to external nucleotides using more purified and more complete complexes or complexes formed from the individual purified enzymes. Never- theless the existence of these enzymes in an aggregate of proteins, the rapid and linear rate of synthesis of product frequently with only a slight lag period in the CDP + dCDP + dCMP + [HmdCMP] sequence, and the resulting low steady state level of the dCMP intermediate, speak to the concept of tightly coupled (42, 43) physically interacting enzymes, and we are in essential agreement with the conclusions of Mathews and coworkers (10, 17, 18).

A salient feature of the T4 deoxyribonucleotide synthetase complex is its sensitivity to salt concentration. The integrity of the complex depends on the presence of relatively low concentrations of salts, and even an increase to 0.09 M brings about partial dissociation to intermediately sized complexes. At 0.2 M KC1 probably all of the enzymes of the complex participating in the formation of deoxyribonucleotides as well as the enzymes of the replication system are dissociated to their free forms (Fig. 7). At the same time the CDP ++ [HmdCMP] pathway was similarly inhibited by these concentrations of KC1 and other salts. This finding needs to be explored further. In a related manner it may be significant to the structure of these complexes that the activity of T4 DNA polymerase is inhibited 97% by 0.2 M KC1 as compared to 0.05

Phage-induced ribonucleoside diphosphate reductase readily dissociates from the complex, even by dilution, and possibly this enzyme is the fiit to be lost from the complex. Naturally this property may have importance in the assembly and regulation of the complex.

This study does not address itself to actual kinetic or functional evidence for the coupling of the deoxyribonucleotide synthetase complex with the replication system. Ongoing studies are directed at this question. However, several earlier findings support the concept that the replication system is physically and functionally associated with the deoxyribonucleotide synthetase complex. The rate of pyrimidine deoxyribonucleotide synthesis in vivo is decreased by about one-third to one-half by amber mutants of gene 43 (DNA polymerase). This effect, clearly not caused by the Dna- state or the resulting accumulation of deoxyribonucleotides since the de- crease is not seen with other Dna- mutants, may represent an interaction between DNA polymerase and the deoxyribonucleotide synthetase complex or one of its components (2,4).

A second and more direct finding that DNA replication is closely coupled to deoxyribonucleotide synthesis derives from our studies in 1976 of infected cells rendered permeable to nucleotides by sucrose plasmolysis (3). In these in situ studies two pathways to phage DNA were found, both replicative and both requiring the products of all of the T4 dna genes, Both pathways were shown to be within each-infected cell. One is the reductive de novo pathway converting ribonucleotides to deoxyribonucleotides and thence to DNA. The second pathway converts deoxyribonucleoside triphosphates to DNA and is dependent on the addition of all 4 of the triphosphates. In the milieu of amino acids and glycerol available in the un- washed preparations as precursors and as an energy source, pathway one forms deoxyribonucleotides. The pathway is blocked by an inhibitor of ribonucleoside diphosphate reductase, forms DNA several times faster than pathway 2, does not require added dNTPs, and in fact is not significantly affected by added dNTP pools. At the same time the simul- taneous synthesis cf dz?xyribonucleotide precursors in the de novo pathway shows L+tlt deviation of the requirement for dNTPs in the dNTP-dependent pathway. That is, the two pathways do not appear to mix, and, therefore, we referred to

M Salt (44).

the de novo pathway as channeled synthesis (3). The de novo pathway also utilizes HmdCMP in the absence of ATP, whereas HmdCTP (or HmdCMP + ATP) enters DNA more slowly by the dNTP-dependent route. This work appears to have been misinterpreted (45). A more recent paper proposing compartmentalization reported that dNMPs (plus ATP) were better substrates than dNTPs in permeable preparations (10) though it is not clear which pathway was being studied (10, 45). Morris and Bittner have suggested that their evidence for two different origins in T4 DNA replication supports the concept of two independent pathways to DNA (46).

In connection with the interaction of T4 DNA polymerase and the deoxyribonucleotide synthetase complex, it is appro- priate to mention again the genetic studies of Chao and coworkers (see “Introduction” (11)). In the same vein the spontaneous occurrence of a second mutation in gene nrdB in an amber mutant of gene 43 (12) may reflect a relationship between these gene products. T4 DNA polymerase is found associated with the deoxyribonucleotide synthetase complex (Figs. 3 and 4 and text to Fig. 5) and appears to be dissociated simultaneously with the enzymes of the complex from the heavy to the lighter fractions and to the free forms (Fig. 7). Taken together with the various observations suggesting that T4 DNA polymerase interacts in vivo with the deoxyribonucleotide synthetase complex, it is attractive to speculate that this enzyme functions as a linker between the complex and the remainder of the replication enzymes (see Refs. 2 and 4).

The occurrence of DNA topoisomerase subunit proteins in association with the deoxyribonucleotide synthetase fractions is particularly intriguing since p39 and p52 are regarded as cytoplasmic membrane proteins (47, 48). By NADH oxidase measurements these fractions (see Fig. 4) appeared to be free of cytoplasmic membrane. If these proteins which are present at high concentrations were carried only by membrane, the complex fractions would be expected to show NADH oxidase activity. The proteins coded by genes rIIA and rIIB, which have been accepted as integral constituents of the cytoplasmic membrane (47, 48), also occur in the deoxyribonucleotide synthetase complex fractions. Thus the proteins coded by 39, 52, rIIA, and rIIB presumably are associated with other proteins in the heavy fractions. Clearly DNA topoisomerase is isolated free of membrane (15, 16).5 This laboratory has found that mutants of the DNA-delay genes coding for the subunits of DNA topoisomerase have inhibitory effects on deoxyribonucleotide synthesis, especially in E. coli S/6 (8), and more recent studies have shown that a defect in the synthesis of a specific nrdB mutant is suppressed by mutants in gene 39.7

The preparations described here are incomplete as compared to the complex defined by our in vivo and in situ studies since as isolated they apparently are neither associated with DNA (6) nor with membrane (8) (see under “Results”). By a variety of procedures we have been able to isolate membrane fractions with many of the deoxyribonucleotide-synthesizing enzymes and replication enzymes still associated? Takacs and Rosenbusch had earlier observed that a number of these proteins were associated with membrane preparations of T4- infected cells, but not as integral proteins (48).

Occurrence of enzymes in a functioning complex is usually considered to provide a means of maintaining higher rates of synthesis of the product (49) and sufficient pools of product to be utilized efficiently by the next enzyme in the sequence.

K. S. Cook, D. 0. Wirak, and G. R. Greenberg, unpublished

‘C.-S. Chiu, K. S. Cook, A. F. Seasholtz, and G. R. Greenberg, results.

unpublished findings.


We have mentioned that in the reaction sequence, CDP + dCDP + dCMP + [HmdCMP], dCMP reaches a low steady state concentration. Because of the limited diffusion requirement, enzyme molecules in close proximity to one another in a complex may allow an intermediate to maintain local concentrations sufficient to approach saturation of the active site of the next enzyme in the sequence. This diffusion advantage is not unlike enzymes in a metabolic sequence immobilized on a surface to form tightly coupled systems in which the factor of proximity and the Nernst diffusion layer form a barrier against outward diffusion and combine to increase the rate of the reaction (50). Indeed, immobilized enzymes in a sequence do not display the lag in the formation of product observed with the individual enzymes in the free state and at the same concentration (50). It may not be simple to distinguish between such a kinetic advantage and a direct transfer from the leaving site of the fiist enzyme to the active site of the next enzyme, facilitated by the juxtaposition of these sites in the complex.

Although it is normally expected that organization of a sequence of enzymes into a complex should lead to a more rapid rate, this conclusion does not follow per se. A common control in a pathway is via feedback inhibitors (or positive effectors) of one of the early irreversible steps (51). In a complex in which regulation depends on protein-protein interactions, the control could lower the rate of an enzymatic step. We have found in in vivo experiments that a mutation in the cd gene coding for dCMP deaminase increased the rate of the deoxyribonucleotide synthetase complex by 1.5 times and have suggested that this could be an effect on ribonucleoside diphosphate reductase (7). This change could not be ascribed to a change in a negative feedback effector since the reductase is sensitive only to positive feedback control (34). It is a curious and as yet unexplained fact that dCMP deaminase, which is very sensitive to feedback inhibition by dTTP (22), does not appear to be inhibited in vivo on infection by Dna- phage (5,7) even though thymidine nucleotides increase to as much as 100 times the wild type level (2).

When this paper was being prepared a related study by Allen et al. appeared. They are in agreement with our earlier arguments and findings suggesting that the deoxyribonucleotide synthetase complex is associated with the replication apparatus (2-4,6,8,13,14) and state that the isolated complex contains T4 DNA polymerase (18). A current abstract reiter- ates this idea (52). These workers suggest that their preparation has been improved by eliminating EDTA before lysis of cells (18). This appears to be an important finding, and we have not addressed this question. Our preparations have al- ways contained T4 DNA polymerase and closely related accessory enzymes in substantial amounts (13, 14). The conditions in our methodology that promote both this association and the apparent lack of association of T4-coded deoxyribonucleoside 5’-monophosphate kinase in the complex remain to be determined. Ionic strength appears as one reasonable candidate.

Deoxyribonucleotide synthetase-like complexes appear to be universal. In recent years complexes carrying several of the enzymes synthesizing deoxyribonucleotides and associated with DNA, membrane, and DNA replication enzymes have been described by Firshein and coworkers in bacteria and other syst.ems (53). Baril et al. have reported such complexes associated with ribonucleotide reductase and other deoxyribonucleotide-synthesizing enzymes and DNA polymerase activity in Novikoff tumor cells (54). Most recently, Reddy and Pardee have separated such a multienzyme complex from a Chinese hamster embryo fibroblast cell line, and, furthermore, these workers have described channeling of ribonucleotides to

DNA in permeabilized cells (55), as Wovcha et al. had found with T4-infected cells (3).

Acknowledgments-We are grateful to Beth Smiley for her excel- lent technical assistance and to our colleagues, Dana Wirak, William Mattes, and Audrey Seasholtz, for productive discussions.

Note Added in Proof-We failed to mention the work of Lunn and Pigiet (56) who found E. coli ribonucleoside diphosphate reductase tightly associated with membrane and who proposed its role in a multienzyme complex.

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11. 12.

13. 14. 15.

16.

17.

18.

19.

20.

21.

22.

23. 24. 25.

26.

27.

28.

29.

30. 31. 32.

33.

34. 35. 36. 37. 38.

REFERENCES Wovcha, M. G., Tomich, P. K., Chiu, C.-S., and Greenberg, G. R.

Tomich, P. K., Chiu, C.-S., Wovcha, M. G., and Greenberg, G. R.

Wovcha, M. G., Chiu, C.-S., Tomich, P. K., and Greenberg, G. R.

Chiu, C.-S., Tomich, P. K., and Greenberg, G. R. (1976) Proc.

Flanegan, J. B., and Greenberg, G. R. (1977) J. Biol. Chem. 252,

Flanegan, J. B., Chiu, C.-S., and Greenberg, G. R. (1977) J. Biol.

Chiu, C.-S., Ruettinger, T., Flanegan, J. B., and Greenberg, G. R.

Wirak, D. O., and Greenberg, G. R. (1980) J. Biol. Chem. 255,

Collinsworth, W. L., and Mathews, C. K. (1974) J. Viol. 13, 908-

Reddy, G. P. V., and Mathews, C. K. (1978) J. Biol. Chem. 253,

Chao, J., Leach, M., and Karam, J. (1977) J. Virol. 24, 557-563 Chiu, C.-S., Cox, S. M., and Greenberg, G. R. (1980) J. Biol.

Chiu, C.-S. (1976) Fed. Proc. 35,1590 Greenberg, G. R., and Chiu, C.-S. (1978) Fed. Proc. 37, 1409 Liu, L. F., Liu, C.-C., and Alberts, B. M. (1979) Nature 281,456-

Stetler, G. L., King, G. J., and Huang, W. M. (1979) Proc. Natl.

Reddy, G. P. V., Singh, A., Stafford, M. E., and Mathews, C. K.

Allen, J. R., Reddy, G. P. V., Lasser, G. W., and Mathews, C. K.

Lomax, M. I. S., and Greenberg, G. R. (1968) J. Bacteriol. 96,

Dale, B. A., and Greenberg, G. R. (1972) J. Bacteriol. 110, 905-

Goulian, M., Lucas, Z. J., and Kornberg, A. (1968) J. Biol. Chem.

Scocca, J. J., Panny, S. R., and Bessman, M. J. (1969) J. Biol.

Warner, H. R., and Lewis, N. (1966) Virology 29, 172-175 Wang, T. (1955) Methods Enzymol. 2, 478-480 Osborn, M. J., Gander, J. E., Parisi, E., and Carson, J. (1972) J.

Yeh, Y.-C., Dubovi, E. J., and Tessman, I. (1969) Virology 37,

Berglund, O., and Sjoberg, B.-M. (1970) J. Bwl. Chem. 245,6030-

Moore, E. C., Reichard, P., and Thelander, L. (1964) J. Biol.

Vogel, H. J., and Bonner, D. M. (1956) J. Biol. Chem. 218, 97-

Laemmli, U. K. (1970) Nature 227,680-685 Cook, K. S., and Seasholtz, A. F. (1982) J. Virol. 42, 767-772 O’Farrel, P. Z., Goodman, H. M., and O’Farrel, P. H. (1977) Cell

Yeh, Y.-C., and Greenberg, G. R. (1967) J. Biol. Chem. 242,1307-

Berglund, 0. (1972) J. Biol. Chem. 247, 7276-7281 Berglund, 0. (1972) J. Biol. Chem. 247, 7270-7275 Schirch, L. (1971) Methods Enzymol. 17B, 330-335 Hibner, U., and Alberts, B. M. (1980) Nature 285,300-305 Cook, K. S., and Greenberg, G. R. (1981) Fed. Proc. 40, 1902

(1973) Proc. Natl. Acad. Sci. U. S. A . 70, 2196-2200

(1974) J. Biol. Chem. 249, 7613-7622

(1976) J. Virol. 20, 142-156

Natl. Acad. Sei. U. S. A. 73, 757-761

3019-3027

Chem. 252,6031-6037

(1977) J. Bwl. Chem. 252,8603-8608

1896-1904

915

3461-3467

Chem. 255,2747-2751

461

Acad. Sci. U. S. A . 76, 3737-3741

(1977) Proc. Natl. Acad. Sci. U. S. A . 74, 3152-3156

(1980) J. Biol. Chem. 255, 7583-7589

501-514

916

243,627-638

Chem. 244,3698-3706

Bwl. Chem. 247,3962-3972

615-623

6035

Chem. 239,3445-3456

106

12, 1133-1142

1313


39. O’Farrell, P. Z., Gold, L. M., and Huang, W. M. (1973) J. Biol. 49. Welch, G. R., and Gaertner, F. H. (1975) Proc. Natl. Acad. Sci.

40. ’Smith, F. L., and Haselkorn, R. (1969) Cold Spring Harbor Symp. 50. Mosbach, K., and Mattiasson, B. (1976) Methods Enzymol. 44,

41. Craine, B. L., and Rupert, C. S. (1978) J. Bacteriol. 134,193-199 51. Yates, R. A., and Pardee, A. B. (1955) J. Biol. Chem. 221, 757- 42. Easterby, J. S. (1973) Bwchim. Biophys. Acta 293,552-558 770 43. Bergmeyer, H. U. (1978) Principles of Enzymatic Analysis, pp. 52. Allen, J. R., Lasser, G. W., and Mathews, C. K. (1981) Fed. Proc.

69-75, Verlag Chemie, Weinheim and New York 40, 1795 44. Kornberg, A. (1980) DNA Replication, p. 187, W. H. Freeman 53. Greene, M., and Firshein, W. (1976) J. Bacteriol. 126, 777-784

and Co., San Francisco 54. Baril, E., Baril, B., Elford, H., and Luftig, R. B. (1973) in Mech- 45. Stafford, M. E., Reddy, G. P. V., and Mathews, C. K. (1977) J. anism and ReguZatwn of DNA Replication (Kober, A. R., and

Virol. 23,53-60 46. Morris, C. F., and Bittner, M. (1981) J. Supramol. Struct. 5,

Kohiyama, M., eds) pp. 275-291, Plenum Publishing Corp., New York

47. Huang, W. M. (1975) Virology 66, 508-521 U. S. A . 77,3312-3316 48. Takacs, B. J., and Rosenbusch, J. P. (1975) J. Biol. Chem. 250, 56. Lunn, C. A., and Pigiet, V. (1979) J. Biol. Chem. 254,5008-5014

Chem. 248,5499-5501 U. S. A . 72,4218-4222

Quant. Bwl. 34,91-94 453-478

(suppl.) 334 55. Reddy, G. P. V., and Pardee, A. B. (1980) Proc. Natl. Acad. Sci.

2339-2350

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