Role of Deoxyribonucleic Acid Ligase in a Deoxyribonucleic

Vol. 126, No. 2

JOURNAL OF BACTERIOLOGY, May 1976, p. 777-784 Copyright © 1976 American Society for Microbiology

Printed in U.S.A.

Role of Deoxyribonucleic Acid Ligase in a Deoxyribonucleic Acid Membrane Fraction Extracted from Pneumococci MILFORD GREENE' AND WILLIAM FIRSHEINI* Department of Biology, Wesleyan University, Middletown, Connecticut 06457 Received for publication 5 February 1976

Deoxyribonucleic acid (DNA) ligase has been detected in a DNA membrane fraction extracted from Pneumococcus. The specific activity of the enzyme in this fraction is 10-fold greater than in the remaining cell extract. It remains firmly bound (with other enzymes) to the complex after a purification procedure in which a considerable percentage of the macromolecules are dissociated. The ligase acts in two ways in the DNA membrane fraction in vitro. One, it catalyzes the linkage of small-molecular-weight pieces of newly synthesized DNA into heavier-molecular-weight DNA strands as shown by others (M. Gellert, 1976; R. Okazaki, A. Sugino, S. Hirose, T. Okazaki, Y. Imae, R. KainumaKuroda, T. Ogawa, M. Arisawa, and Y. Kurosowa, 1973; B. Olivera and I. Lehman, 14; and A. Sugino, S. Hirose, and R. Okazaki, 1972) and, two, it protects DNA from degradation by deoxyribonucleases. This latter effect is due to a competition between the ability of the nucleases to degrade DNA and the ability of DNA ligase to seal the nicks produced by these degradative enzymes. The ligase acts cooperatively with other enzymes in the DNA membrane fraction to synthesize DNA. MATERIALS AND METHODS Organism, preparation of cell suspensions, and extraction of DNA membrane fraction (i). The organism (an encapsulated strain of type III Streptococcus pneumonzae), preparation of cell suspensions to obtain "preparative" amounts of the DNA membrane fraction, the use of a supplement to enhance DNA synthesis in the cell suspensions, and the extraction of a DNA membrane fraction by the use of the detergent sodium lauroyl sarkosinate (Sarkosyl) were all described in detail previously (4-6). Macromolecular analysis (ii). Bulk DNA and protein were measured by the method of Burton (1) and Lowry et al. (11), respectively. Nascent DNA synthesis in the DNA membrane fraction was measured by pulsing cell suspensions with radioactive DNA precursors for 30 or 120 s. Ice-cold TMK buffer {tris(hydroxymethyl)aminomethane [Tris], 0.01 M; 0.1 M KCl, 0.02 M magnesium acetate, final pH 7.4} containing sodium cyanide (0.02 M, final concentration) was added to stop the reaction. The cell suspensions were washed three times with TMK buffer without cyanide, treated with Sarkosyl to extract the DNA membrane fraction, and used as a substrate for DNA ligase activity (see below). Assay for DNA ligase activity in the DNA membrane fraction (iii). Bacterial DNA ligase requires nicotinamide adenine dinucleotide (NAD) as a cofactor (14) and can be measured in a variety of ways. of these were used in this investigation. Three Uniof Biochemistry, 'Present address: Department Method (i) involves the detection of nicotinamide versity of Massachusetts School of Medicine, Worcester, mononucleotide (NMN) from NAD as one end proMass. 01605.

A deoxyribonucleic acid (DNA) membrane fraction extracted from pneumococci was found to contain a DNA replication complex (2, 4). A number of enzymes were detected in this fraction which acted cooperatively in synthesizing DNA, including deoxyribonucleoside monoand diphosphate kinases, DNA polymerase, ribonucleoside diphosphate reductase, and DNA ligase. The detection of this latter enzyme was considered of utmost importance since, according to a number of studies (9, 13), DNA synthesis occurs in a discontinuous manner. Both strands are replicated in the 5'-3' direction as short fragments and then linked by DNA ligase to form the long strands of chromosomal DNA. That this process may have occurred in the DNA membrane fraction in vivo was seen by the detection of such newly synthesized DNA fragments (sedimenting at 9-11S in an alkaline sucrose gradient) after extraction of the complex from cells pulsed for a short time with a labeled DNA precursor (2). This report analyzes further the role of DNA ligase in the DNA membrane fraction of pneumococci and indicates another function of the enzyme in addition to its joining activity, that of an indirect inhibitor of DNase activity.

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duct of the reaction. Method (ii) involves an analy- ml, final concentration) for 10 min at 37 C and diasis of the molecular size of single-stranded DNA in lyzed for 5 h at 4 C against the same buffer used to alkaline sucrose gradients extracted from cells remove Sarkosyl and other components from the pulse labeled with [3H]deoxycytidine (2) and in- DNA membrane fraction (Tris, glycerol, albumin; cubated with or without NAD. Method (iii) meas- 0.01 M, 20%, and 1.0 mg/ml, respectively, pH 7.5), ures the cooperative effects of NAD on in vitro DNA and 0.7 ml of the dialyzate was used to assay for synthesis of the DNA membrane fraction. ligase activity in the presence of [3H]NAD or For method (i), the incubation mixture (0.88 ml) [3H]NAD + NMN. There was an 85% decrease in contained [3H]NAD (1.4 x 10-6 M) (104 counts/min the amount of diphenylamine-positive material (1) per umol), NMN (2.9 x 10-7 M), potassium phos- in the dialyzed DNA membrane fraction but little phate buffer, pH 6.5 (0.05 M), MgCl2 6H2O (0.01 or no decrease in the amount of protein as judged by M), and DNA membrane or top fraction (the cell the Folin-phenol reagent assay (11). A similar treatextract remaining after extraction of the DNA mem- ment of the DNA membrane fraction without brane fraction) (0.7 ml of each fraction). The DNA DNase resulted in a 25% decrease of diphenylaminemembrane fraction contained 80 ,ug of DNA and 390 positive material when compared with the unjig of protein per 0.7 ml, whereas the top fraction treated fractions, presumably due to endogenous contained 14 ,ug of DNA and 640 Ag of protein per DNases present in the DNA membrane complex. 0.7 ml. The DNA concentration of the latter was (See Fig. 1A, B). made equivalent to the former by adding an apAfter 25 min of incubation at 37 C, the samples propriate amount of DNA extracted from a replicate were heated for 1 min at 100 C and chilled in ice, sample of the DNA membrane fraction as described and marker nucleotides (0.02 ml of 5 mM NAD and 1 by Marmur (12). In one case, the DNA membrane mM NMN) were added. The entire mixture was apfraction was treated with pancreatic DNase (0.1 mg/ plied to chromatographic paper (Whatman no. 1). 14S 12S

14S 12S 1200 .2S

A. 30 sec.

z E

I

1000

A:

w z

x B. 120 sec.

I

\

\

I~~~~~

400

10

14

18

22 4-TOP

14

18

22 4-TOP

FRACTION NO. FIG. 1. Alkaline sucrose gradient centrifugation of pulse-labeled DNA in the DNA membrane fraction. Cell suspensions were pulsed for 30 or 120 s with [3H]deoxycytidine (1 uCiIml) and, after extraction of the DNA membrane fraction, two equivalent portions were incubated for 20 min with and without NAD, while a third portion was not incubated. All three samples were layered on top of an alkaline sucrose gradient and centrifuged as described.. Sedimentation values were calculated by comparison to bacteriophage A DNA marker molecules. To determine whether there was a greater percentage of single-stranded DNA with a larger molecular weight in the NAD-augmented assays than in both controls (unsupplemented and incubated or not incubated), the total amount of radioactivity was determined in each peak (fractions 19 to 24 from A; and fractions 17 to 24 from B), and the percentage of DNA in the former sample sedimenting at sedimentation values greater than the peak values ofboth latter controls was calculated. Symbols: i, radioactivity ofDNA in DNA membrane fraction incubated with NAD; 0, radioactivity of DNA in DNA membrane fraction incubated without NAD; x, radioactivity of DNA in DNA membrane fraction unincubated.

VOL. 126, 1976

DNA LIGASE ACTIVITY IN MEMBRANE COMPLEX

The paper was developed for 15 h by descending chromatography with solvent C of Preiss and Handler (15) modified slightly to contain seven parts of 95% ethanol to three parts of 1 M ammonium acetate buffer, pH 4.8. Rf values in the system were: NAD, 0.47; NMN, 0.79; nicotinamide, 0.95. NAD and NMN spots were located by quenching of ultraviolet light, cut out, and counted in the Nuclear-Chicago scintillation counter (Mark I) using a toluene base scintillation fluid. The recovery of added counts in the two spots was greater than 85% in all cases. If the enzyme (either fraction) was omitted, 7% of the tritium was found in the NMN spot; this value has been subtracted frQm all data reported in the results. In method (ii), pulse-labeled DNA in a DNA membrane fraction was incubated in the presence of NAD using the same final volume (0.3 ml) and concentration of NAD (3.2 ,umol/0.01 ml), MgCl (7 mM), and DNA membrane fraction (25 ug of DNA/0.29 ml of dialyzate buffer) as in method (iii) (see below). No other substrates or cofactors were added. After incubation for 25 min at 37 C, the molecular size of single-stranded pulse-labeled DNA was determined in the following way (controls which were either incubated without NAD or not incubated were also analyzed). Each solution was placed in ice and sodium hydroxide (0.05 M) (0.03 ml) was added to denature the DNA and dissolve membranes. After standing for a few minutes, the incubation mixture was carefully layered onto 34 ml of a 40 to 60% (wt/vol) alkaline sucrose gradient (0.9 M NaCl, 0.1 M NaOH, 1 mM ethylenediaminetetraacetic acid) over 6 ml of an 80% sucrose shelf and centrifuged for 12 h at 22,000 rpm in an SW27 rotor in a Spinco L-2 ultracentrifuge. Fractions were collected (2.0 ml) and an equal volume of cold 10% trichloroacetic acid was added to each fraction. The precipitate was allowed to stand in ice for 30 min and then poured onto a membrane filter (0.45-j,m HA; Millipore Corp.). The filters were washed twice with 10-ml portions of cold 5% trichloroacetic acid and once with 10 ml of cold water, dried for 2 h at 60 C, and then counted as described above. The percentage of DNA that shifted to a heavier singlestranded molecular weight in the presence of NAD from the peak value of the DNA sedimenting in the alkaline gradient in the absence of NAD or in the unincubated control was taken as a measure of ligase activity (see legend of Fig. 1). An internal marker of X bacteriophage DNA was used in the gradients. In method (iii), DNA synthesis in the DNA membrane fraction was assayed in vitro in the presence and absence of NAD (3.2 nmol/0.01 ml, see below). The complex was first dialyzed against the buffer described above (Tris, 0.01 M, 20% glycerol, and 1 mg of bovine serum albumin per ml (fraction V), final pH 7.4) for 12 h at 4 C. To samples of the dialyzate, the following were added (total, 0.3 ml): Tris (0.2 M, pH 8.6) (0.03 ml); thymidine 5'-triphosphate, deoxycytidine 5'-triphosphate (dCTP), deoxyguanine 5'-triphosphate (dGTP) (0.04 Amol) (0.02 ml); [8-14C]deoxyadenosine 5'-triphosphate (dATP) (6.5 x 106 counts/min per ,umol) (0.04

779

,umol) (0.02 ml); MgCl, 7 mM (0.01 ml); dithiothreitol, 1 mM (0.02 ml); DNA membrane fraction adjusted to 25 lAg of DNA per 0.15 ml in dialysis buffer. This was the complete system for deoxyribonucleoside triphosphate-initiated DNA synthesis (with or without NAD). The complete system for the assay of deoxycytidylate (dCMP)- or deoxycyti-

dine 5'-diphosphate (dCDP)-initiated DNA synthesis consisted of the same as described above except that [2-14C]dCMP (106 counts/min per ,umol) (0.08 ,umol) (0.02 ml) or [2-14C]dCDP (2.1 x 106 counts/min per ,umol) (0.08 ,umol) (0.01 ml) was substituted for dCTP. In addition adenosine 5'-triphosphate (2.0 ,umol) (0.01 ml), creatine phosphate (8 jAmol) (0.01 ml), and creatine phosphokinase (10 jAg) (0.01 ml) were added and ['4C]dATP was replaced by nonlabeled dATP. Other conditions were the same. After incubation for 25 min at 37 C, each assay solution was placed in ice, an equal volume of cold 10% trichloroacetic acid + 0.01 M sodium pyrophosphate was added, and the precipitate was allowed to stand in ice for 30 min. Acid-insoluble radioactivity was determined as described in method (ii) except that 0.01 M sodium pyrophosphate was added to all of the trichloroacetic acid washes. Purification of DNA membrane fraction (iv). By a combination of CsCl and sucrose gradient centrifugation, it was possible to remove a considerable percentage of macromolecules from the DNA membrane fraction without impairment of in vitro DNA synthesis. The method was described in detail

previously (2). Effect of NAD on degradation of pneumococcal DNA by pneumococcal deoxyribonucleases (v). These assays were performed as described previously (4). Labeled pneumococcal DNA was prepared as described by Marmur (12) and diluted to contain the same amount of radioactivity present in the experiments described in Fig. 1A and B. Pneumococcal DNases were extracted as described by Lacks and Greenberg (10). The nucleases were not separated, so that the state of purification was approximately sevenfold from the crude deoxycholate extract (10). NAD was added to the assay solution at a concentration of 2.5 ,ug/0.5 ml. DNase activity was expressed as the percentage of acidinsoluble radioactivity in DNA rendered acid soluble after various incubation times compared with control assays that were not incubated but treated immediately with cold trichloroacetic acid after preparation. Radioactive and other substrates (vi). All radioactive DNA precursors and NAD were purchased from Schwarz/Mann. These included the four 14Clabeled precursors for DNA: [2-'4C]deoxycytidine,

[8-'4C]deoxyadenosine triphosphate, [2-14C]-deoxycytidine monophosphate, and [2-'4C]deoxycytidine diphosphate (their specific activities and concentrations are described in other sections of the Materials and Methods). All nonlabeled compounds including polyadenylic acid and all eight of the naturally occurring deoxyribonucleosides and -tides (which are used in the

780

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GREENE AND FIRSHEIN

cell suspensions to enhance DNA synthesis in pneumococci) (5), their triphosphate derivatives, ATP, creatine phosphate and its kinase, NAD, NMN, and nicotinamide were obtained either from Sigma Chemicals or Calbiochem.

RESULTS

DNA ligase in the DNA membrane and top fraction. The presence of DNA ligase in the DNA membrane fraction was indicated by previous results with alkaline sucrose gradient centrifugation of newly synthesized DNA (extracted from the DNA membrane fraction in vivo) (2). With increasing pulse times beginning at 15 s, the sedimentation of denatured labeled DNA in the sucrose gradient increased from 9-11S to 16-20S after 180 s. These results suggested that DNA was synthesized in small pieces and subsequently linked to heaviermolecular-weight DNA, presumably by DNA ligase activity in vivo. To specifically detect DNA ligase activity in vitro and to determine its distribution in the total cell extract, the production of [3H]NMN from [3H]NAD was measured after incubation of the DNA membrane and top fractions. In addition, some of the assays were performed in the presence of an excess of NMN to ascertain more fully the role of NAD as a specified cofactor for DNA ligase in pneumococci. The possible contamination of another enzyme that degrades NAD to NMN (nucleotide pyrophosphatase) (17) was assessed by assaying the DNA membrane fraction for the conversion of NAD to NMN after removal of DNA by pancreatic DNase as described. If the above phosphatase were present, the removal of most of the DNA should not affect its activity and similar levels of [3H]NMN should be produced from the [3H]NAD substrate. In contrast, if NAD is acting primarily as a cofactor for DNA ligase, the removal of DNA should affect its activity adversely. Percentage of conversion of substrate ([3H]NAD) to product ([3H]NMN) per milligram of protein in the DNA membrane and top fractions was used to determine specific activity. The amount of NMN formed from NAD was proportional to the amount of DNA membrane complex added over a sixfold range of concentration. This same method of determining the product to substrate ratio in other enzymatic assays was reported previously (2). It can be seen from the results of Table 1 that, when the DNA membrane or top fraction extracted from pneumococci was incubated in the presence of [3H]NAD, the specific activity of DNA ligase was greater in the DNA membrane fraction than in the top fraction (42

TABLE 1. In vitro DNA ligase activity of the DNA membrane and top fractiona Addition to in vitro

system

Sp act

product/substrate per

mg of protein

DNA membrane

[3H]NAD [3H]NAD + NMN

42.4 27.1

DNA membrane pretreated with DNase Top

[3H]NAD [3H]NAD + NMN [3H]NAD [3H]NAD +

6.5 3.8 4.1 4.3

NMN The specific assay for in vitro DNA ligase activity is described in Materials and Methods. When enzyme (DNA membrane fraction or top fraction) is omitted, the value of P/S is zero. Protein was determined by the method of Lowry et al. (11). a

versus 4.1) despite the presence of equivalent levels of DNA. (Total enzymatic activity was also greater despite the presence of only 35% of the total cell protein in the DNA membrane complex, not shown.) Furthermore, the addition of NMN to the reaction mixture shifted the equilibrium to the left, with a corresponding decrease in specific activity of the M-band fraction. In contrast, the addition of NMN to the reaction mixture had little or no appreciable effect upon the specific activity of the enzyme in the top fraction, suggesting that ligase activity was not present at all in this fraction. When the assay was performed on a DNA membrane fraction pretreated with DNase, there was a drastic inhibition of the conversion of NAD to NMN (almost sevenfold). The remaining activity was probably not due to phosphatase activity but to ligase activity on relatively large polydeoxynucleotides that were not degraded by DNase and hence not removed after dialysis of the DNA membrane fraction. Thus, it seems (i) that NAD is a cofactor for DNA ligase activity in pneumococci and (ii) that the specific (and total) activity of the enzyme is greater in the DNA membrane fraction than in the remaining cell extract. Further evidence for the presence of ligase activity as a function of NAD is described below. Effects of NAD on the molecular size of pulse-labeled DNA in the DNA membrane fraction. To ascertain whether newly synthesized DNA in the DNA membrane fraction would be affected by the addition of NAD, the following experiment was performed. The DNA membrane fraction was extracted from cells pulselabeled with [3H]deoxycytidine for 30 or 120 s, either not incubated further or incubated with

VOL. 126, 1976


781 and without NAD, and centrifuged in a linear Effect of NAD on DNase activity. To dealkaline sucrose gradient as described (Fig. termine whether NAD affects the degradation

1A, B). It can be seen that before incubation, the 30-s pulse-labeled denatured DNA sedimented in a broad peak with an average sedimentation rate of 12S (Fig. 1A), whereas after 120 s the DNA sedimented at slightly heavier sedimentation values (Fig. 1B). When the DNA membrane fraction containing 30- or 120-s pulse-labeled DNA was incubated for 20 min without NAD, both DNA samples also sedimented in broad peaks of about 12S or slightly heavier sedimentation values, respectively, but the amount of DNA detected on the gradients was approximately 40 to 50% less than that detected before incubation (Fig. 1A, B). In contrast, when each pulse-labeled DNA membrane fraction was incubated with NAD, not only was there a much greater percentage of denatured DNA retained on the gradients than in the unsupplemented incubated samples, but there was a significant increase in the molecular size of the single-stranded DNA. This was particularly noticeable in the 120-s pulse-labeled material in comparison with both the unincubated and incubated unsupplemented controls (Fig. 1B). Approximately 22 and 30%, respectively, of the DNA obtained from the 120-s pulse-labeled DNA membrane fraction incubated with NAD sedimented at sedimentation values greater than the average value for the two controls. These results suggest that the process of joining short DNA fragments can be detected in vitro and that the reaction is mediated by DNA ligase because (i) NAD was required for the observed activity and (ii) the only enzymatic way in which an increase in the molecular size of single-stranded DNA could occur in the NADaugmented samples as compared with the unincubated controls would be by a covalent joining of DNA fragments. However, another effect of NAD and/or DNA ligase activity is seen that might involve the inhibition of DNase activity since a considerable percentage of the DNA was degraded after incubation of the DNA membrane fraction for 20 min without NAD. In contrast, a much smaller percentage of such DNA was degraded when NAD was present. Pneumococcus is known to possess both endoand exonucleases (10), and we have detected exonuclease activity in the DNA membrane fraction (4). The main alternative to the action of DNA ligase in the manner described would be that NAD affects DNase directly either as an inhibitor or as a protective agent for its substrate, DNA. This would produce results that resemble ligase action. Such a possibility is examined below.

of pneumococcal DNA by a mixture of pneumococcal DNases, experiments were performed in which NAD was added directly to assay solutions containing DNA and DNase as described previously (4) and in Materials and Methods. The amount of DNA used in this experiment was comparable to that used in Fig. 1 with respect to specific activity of the labeled DNA. The results demonstrate (Fig. 2) that NAD does not affect the degradation of DNA by the DNase preparations used. As can be readily seen, there was no appreciable change in the percentage of DNA degraded to acid-soluble material when NAD was added to the reaction mixture. Thus, we conclude that the increased level of DNA detected in the alkaline gradients in the presence of NAD (as shown in Fig. 1A,B) is due to the action of DNA ligase which counteracts the effects of DNases. DNA synthesis in vitro in various fractions during purification of the DNA membrane fraction. A procedure was developed to purify the DNA membrane fraction with respect to its ability to synthesize DNA in vitro. It was described in detail previously (2). A determination was made of the ability of the various 1001_ w m J -J

0 Uf) 0

8OF

w 60 _ a

--

8

0

z 40~ a

z

U')

I-

z w 201 C.) w a. I

I

10

20

I

I

I

40 50 30 TIME (MIN) FIG. 2. Effect of NAD on the degradation of pneumococcal DNA by a preparation of pneumococcal DNases. Assay mixtures and other conditions for determining the effect of NAD on the degradation of DNA by DNase are described in Materials and Methods. Symbols: *, with NAD; 0, without NAD.

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fractions to synthesize DNA alone and in the of NAD (Table 2). It can be seen that, after dialysis and centrifugation in CsCl, the DNA membrane fraction (phospholipid pellicle) remaining at the meniscus retained about 80% of the total DNA synthetic activity initiated by deoxyribonucleoside triphosphates in the absence of NAD. There was approximately 80% retention of the total activity measured in the presence of NAD. When the rebanded phospholipid pellicle from the sucrose gradient was assayed, total triphosphate-initiated DNA synthesis declined substantially to about 33% of the original total activity in the absence of NAD. Again, however, the addition of NAD allowed for the retention of more activity, 45% of the total remaining. Although there was a reduction in total enzyme activity, the specific activity increased in either system (with or without NAD) after each purification step. There was no specific measurement of whether the molecular size of the newly synthesized DNA was greater in the presence of NAD than in its absence during each purification step. However, the retention of a greater level of synthetic activity in the presence of the ligase cofactor suggested that the molecular size of the synthetic product was also greater, since the results of Fig. 1A and B demonstrated in vitro that both events were coupled. Thus, these data demonstrate that DNA ligase "cooperates" with DNA polymerase in the synthesis of DNA by protecting the substrate against degradation and presumably linking the newly synthesized DNA fragments to form the longer strands of chromosomal DNA. Furthermore, certain treatments can dissociate various amounts of macromolecules from the DNA membrane fraction without destroying this physiological activity. Cooperative effects between DNA ligase and various enzymes in the purified DNA mem-

presence

brane fraction. Previous results (4) showed that DNA ligase "cooperated" with other enzymes in minced gel extracts after electrophoresis of the DNA membrane fraction in polyacrylamide gels. Higher levels of DNA were synthesized when NAD was added to assay solutions containing substrates for deoxyribonucleotide kinase and DNA polymerase activity. That such cooperative effects still occurred with fraction III of the purified DNA membrane fraction (see Table 2) was shown by the following results (Table 3). When dCTP was replaced either by dCDP or dCMP plus the necessary cofactors for kinase activity and the remaining complete system for triphosphate-initiated DNA synthesis (see Materials and Methods), the specific activity (counts per minute of acid-insoluble material per milligram of protein) was 670 and 1,400, respectively. When NAD was added, the specific activity increased to 1,670 and 2,800, respectively. It is interesting to note that DNA synthetic activity was greater when it was initiated by dCMP than dCDP despite the fact that dCDP is a closer intermediate to the end product (DNA) than dCMP. This result may be related to the catalytic facilitation of the multienzyme complex involved in DNA synthesis in which early substrates are more efficient than later ones, as shown by other investigators studying multienzyme complexes (6). We conclude that DNA ligase remains firmly bound with other enzymes to the DNA membrane fraction and acts cooperatively as part of a multienzyme complex in this fraction to synthesize DNA. DISCUSSION The detection of DNA ligase activity in a DNA membrane complex extracted from pneu-

TABLE 2. DNA synthesis in vitro in the presence and absence of NAD during purification of the DNA membrane fractiona act without with Sp Sp act(counts/ without NAD Fraction Total activity Total activity NAD (counts/ NAD

Fractionwi~thouta NADvt FractionchountsinA

I. DNA membrane fraction extracted by Sarkosyl

II. Dialysis and centrifugation in CsCl gradient and then dialysis of the DNA phospholipid pellicle

III. Sucrose gradient centrifuga-

with NAD

min per mg of protein)

29,250

33,600

450

500

20,500

27,950

700

950

6,750

11,850

1,950

3,400

min per mg of protein)

tion and rebanding a Assay mixtures for DNA synthesis initiated with deoxyribonucleoside triphosphates and other conditions were described in Materials and Methods. Purification of DNA membrane fraction was described previously (2). Protein was determined by the method of Lowry et al. (11).

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783

TABLE 3. DNA synthesis by purified fraction III in vitro using various substrate combinations Sp act (counts/min Activities measured Conditionsa per mg of protein) DNA polymerase DNA polymerase + DNA ligase

Complete (all 4 triphosphates) Comnplete + NAD

Diphosphate kinase + DNA polymerase Same as above + DNA ligase

-dCTP + [14C]dCDP + ATP -dCTP + ['4C]dCDP + ATP + NAD

1,95(Y 3,400 670 1,670

1,400 -dCTP + [14C]dCMP + ATP Monophosphate kinase + diphosphate kinase + DNA polymerase 2,800 -dCTP + [14C]dCMP + ATP + NAD Same as above + DNA ligase a Fraction III of Table 2 was supplemented with the various substrates and cofactors for detecting the sequential action of kinases and DNA polymerase in synthesizing DNA as described in Materials and Methods. The addition of NAD tested the cooperative effects of DNA ligase with these other enzymes. b Taken from Table 2, fraction Ill.

mococci adds considerable importance to the complex as the structural unit for DNA replication in vivo. This is due to the fact that DNA ligase is an essential enzyme whose action is required for normal DNA replication according to many models now proposed (8, 13, 14, 16). DNA is synthesized discontinuously on one or both strands and an absolute requirement is that the discontinuous fragments of newly synthesized DNA are linked together by DNA ligase. The previous detection of small pieces of newly synthesized DNA in the DNA membrane fraction supports the idea that discontinuous DNA replication occurs in the complex (2). Thus far, we have detected all four deoxyribonucleotide kinases, nucleoside diphosphate kinase, ribonucleoside diphosphate reductase, at least two DNA polymerases, ribonucleic acid polymerase, and DNA ligase in the DNA membrane complex (2, 4). The fact that most of these enzymes remain firmly bound to the DNA fraction during two different purification procedures during which different percentages of macromolecules are dissociated suggests the presence of a multienzyme complex cooperating in the synthesis of DNA. The reaction with DNA ligase is of particular interest because it may have another function in addition to catalyzing the linkage of DNA intermediates into the long strands of chromosomal DNA, that is, as an indirect inhibitor of DNase activity. In every case where DNA ligase activity was activated in vitro by the addition of its cofactor NAD, the amount of DNA detected was greater than in its absence (see Tables 2, 3; Fig. 1A and B). It appears that DNA ligase competes with the degradative effects of DNase by sealing the initial nicks made by these latter enzymes. Although

Ganesan (7) found in studies with a DNA polymerase complex derived from Bacillus subtilis that once the DNA product was made DNA ligase protected it from nuclease action as in the present studies with pneumococci (Fig. 1A and B), he also found, in contrast, that the activation of DNA ligase during the DNA polymerase reaction in vitro inhibited DNA synthesis. These latter results are opposite to those obtained with pneumococci as shown in Tables 2 and 3. One possible explanation for the results of Ganesan (7) was that, by linking together newly synthesized DNA fragments through DNA ligase action, fewer initiation sites (in the form of open 3'-OH- groups) would be available for DNA polymerase activity. If this is correct, the difference in our results could be due to the fact that the rate of DNA polymerase activity is faster than the rate of DNA ligase activity for a number of reasons. These include (i) the relative amounts of each enzyme in the complex, (ii) the amount of NAD added, (iii) control factors present in the complex extracted from pneumococci that compensate for the inhibitory effect seen by Ganesan (7), and (iv) other nonspecific factors such as the assay solution, etc., which might not be as good for detection of ligase action as for DNA polymerase activity. Since DNA ligase is an essential enzyme involved in DNA replication and presumably must act in concert with other enzymes, our stimulatory results suggest an explanation in which natural mechanisms are operative such as those described in (i) and (iii) above. Finally, it is interesting that the specific activity of DNA ligase in the DNA membrane fraction is 10 times that found in the remaining cell extract (see Table 1). Gottesman et al.

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GREENE AND FIRSHEIN

(9) found that ligase normally exists in vast excess in Escherichia coli, since cells were insensitive to large changes in ligase levels. However, if this is so, there should be no reason to find an enrichment of ligase activity in the DNA membrane fraction. Such an enrichment suggests, at least in Pneumococcus, a limited supply of ligase molecules which must be concentrated in the microenvironment in which they act. ACKNOWLEDGMENT This research was supported in part by National Science Foundation grant GB34155. 1.

2. 3.

4.

5.

6.

LITERATURE CITED Burton, K. 1956. A study of the conditions and mechanisms of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. J. 62:315-322. Firshein, W. 1972. The DNA-membrane fraction of Pneumococcus contains a DNA replication complex. J. Mol. Biol. 70:383-397. Firshein, W. 1965. Influence of deoxyribonucleic acid degradation products and orthophosphate on deoxyribonucleotide kinase activity and deoxyribonucleic acid synthesis in Pneumococcus type III. J. Bacteriol. 90:327-336. Firshein, W. 1974. In situ activity of enzymes on polyacrylamide gels of a deoxyribonucleic acid-membrane fraction extracted from pneumococci. J. Bacteriol. 118:1101-1110. Firshein, W., and R. Benson. 1968. Effects of polyribonucleotides of known composition on deoxycytidylate and deoxyguanylate kinase activity in pneumococci. J. Biol. Chem. 243:3301-3311. Gaertner, F. H., M. C. Ericson, and J. A. De-

J. BACTERIOL.

7. 8.

9.

10. 11.

12. 13.

14.

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