Proc. Nat. Acad. Scd. USA Vol. 73, No. 4, pp. 1053-1057, April 1976 Biochemistry Involvement of Escherichia coli dnaZgene product in DNA elongation in vitro [bacteriophage qX174, fd, and ST-1 DNAs/DNA nucleotidyltransterases (polymerases;) II and III/DNA replication] SUE WICKNER* AND JERARD HURWITZt * Laboratory of Molecular Biology, National Institute of Arthritis, Metabolism and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20014; and t Department of Developmental Biology and Cancer, Division of Biological Sciences, Albert Einstein College of Medicine, Bronx, New York 10461 Contributed by Jerard Hurwitz, January 8, 1976

ABSTRACT E. coli dnaZ gene product is required for Schekman et al. (15) have found the conversion of G4 conversion of OX174, fd, and ST-i single-stranded phage DNA to RFII requires dnaG gene product, DNA binding DNAs to duplex DNAs in vitro. This protein has been puri- fied about 5000-fold. It functions in the elongation of RNA- protein and DNA polymerase III holoenzyme. The protein or DNA-primed single-stranded DNA that is catalyzed by requirements we observe for this reaction dependent on'G4, DNA polymerase III (DNA nucleotidyltransferase; deoxynu- St-i, and 4XtB DNA include dnaG gene product, DNA cleosidetriphosphate: DNA deoxynucleotid ltransferase; EC binding protein, DNA polymerase III, and DNA elongation 2.7.7.7) in conjunction with two other E. coli protein prepara- factors I and 11 (5). tions referred to as DNA elongation factors I and III. It also We have found that the conversion of qX174 DNA to functions in similar reactions catalyzed by DNA polymerase II in combination with E. coli DNA binding protein and RFII requires four dNTPs, Mg+2, ATP, and at least 11 puri- DNA elongation factors I and III. fied protein fractions: dnaB, C(D), and G gene products, DNA binding protein, DNA polymerase II, DNA elongation Genetic studies of Escherichia coil mutants temperature factors I and II, and four other E. coli protein preparations sensitive (ts) for DNA synthesis have resulted in the identifi- which are as yet genetically undefined [referred to as DNA cation of genes whose functions are required for chromo- replication factors X, Y,'and Z (16)]. Schekman et al. (15) some replication. These loci have been designated dnaA, B, have reconstituted this system with eight protein prepara- C(D), E, G, H, I, P. and Z, lig and polAexl (see ref. 1). tions; DNA synthesis requires four NTPs, four dNTPs, Some of these E. coli proteins are also required for the con- Mg+2, and spermidine. version of single-stranded circular phage DNAs to duplex We report here the requirement for dnaZ gene product in DNA, catalyzed by crude extracts of uninfected E. coli. fd all three of these DNA synthesizing systems and show its in- (M13) DNA synthesis requires dnaE gene product (2) and volvement in the elongation of primed single-stranded RNA polymerase (3) but not dnaA (4), dnaB (4), dnaC(D) DNA. We have found that DNA elongation factor II prepa- (2), or dnaG (2); G4 DNA synthesis [also ST-1 and 6XtB rations consist of dnaZ gene product plus another protein DNAs (5)] requires dnaC but not dnaB or C(D) gene prod- factor we will refer to as DNA elongation factor III. ucts or RNA polymerase (6); and qX174 DNA synthesis re- quires dnaB (7), dnaC(D) (2), dnaE (DNA polymerase III) MATERIALS AND METHODS (2), and dnaG (2) gene products but not dnaA (7) or RNA polymerase (3). Using crude extracts of E. coli dna mutants Materials, Reagents, and Methods, unless otherwise in- in in vitro complementation assays for these proteins, we dicated, were as previously described (8). E. coli strains used have isolated and partially characterized dnaB, C(D), and G included AX727 (dnaZ ts) and AX729 (dnaZ+) (1); BT1040 gene products (8, 9); recently dnaG product has been isolat- (dnaE ts), BT1026 (dnaE ts), E4860 (dnaE ts), and NY73 ed by others (6). (dnaG ts) (17). ST-1 phage was kindly given by C. Dowell; Each of the above DNA synthesizing systems has been re- E. coli strains AX727 and AX729 were kindly given by J. constituted with purified protein fractions. The conversion Walker. of fd DNA to replicative form II (RFII) requires Mg 2, four Preparation of Proteins. E. coli DNA binding protein NTPs, four dNTPs, RNA polymerase, DNA binding protein, and DNA elongation factor I were purified from E. coli DNA polymerase III (DNA nucleotidyltransferase; deoxynu- strain NY73, DNA polymerase III from NY73, DNA poly- cleosidetriphosphate:DNA deoxynucleotidyltransferase; EC merase II from BT1040, and DNA polymerase I from E4860 2.7.7.7), and DNA elongation factors I and 11 (10-12). Fur- by procedures modified from those in refs. 18, 11, 19, 20, thermore, we have found that any long single-stranded and 21, respectively. The assay conditions and definition of DNA primed with RNA or DNA can be elongated by DNA units for DNA polymerase III are in ref. 10; those for other polymerases II or III in combination with DNA elongation enzymes are given in the above references. The procedure factors I and II (11). W. Wickner and Kornberg (13, 14) for isolation of DNA elongation factor III will be published have reported that this reaction can be catalyzed by DNA elsewhere. polymerase III holoenzyme or by DNA polymerase HII* plus In Vitro Complementation Assays for dnaZGene Prod- copolymerase III*. We presume that DNA elongation factor uct. Reaction mixtures (0.03 ml) for 4X174 and ST-1 DNA- I and copolymerase III* are the same protein and DNA dependent activity contained 20 mM Tris-HCl (pH 7.5), 10 polymerase III plus DNA elongation factor II are operation- mM Mg9l2, 4 mM dithiothreitol, 3 mM ATP, 0.04 mM each ally the same as DNA polymerase III*. of dATP, dGTP, dCTP, and [3H]dTTP (500-1000 cpm/ pmol), 10 tig/ml of rifampicin, 300 pmol of .X174 or ST-1 Abbreviations: RFII, double-stranded DNA of circular replicative DNA, 0.5 mg of ammonium sulfate fraction prepared from form with a discontinuity in at least one strand; ts, temperature sen- dnaZ ts cells (8), and protein fractions containing dnaZ gene sitive. product. Reaction mixtures for fd DNA-dependent activity 1053 1054 Biochemistry: Wickner and Hurwitz Proc. Nat. Acad. Sci. USA 73 (1976)

100 Ad B '25X > 60 F4040 ~ 0 4 >i49 20 20 15- p 10 aA a An10 I. z 0 0. 5 0. 2

5 10 15 5 10 15 TIME AT 39- (minutes) TIME AT 37- (minutes) 2 4 6 8 10 VOLUME OF EXTRACT (1i) FIG. 1. Heat inactivation of 4X174 (A) and ST-1 (B) DNA- dependent DNA synthesis in extracts of dnaZ ts (AX727) and FIG. 2. fd DNA-dependent activity in extracts of dnaZ tb dnaZ+ (AX729) strains. Extracts of each strain were prepared (8) (AX727) and dnaZ+ (AX729) strains. Reaction mixtures were as and incubated as indicated. The other components of the com- described in Materials and Methods with dnaZ gene product plementation assays except purified dnaZ gene product were omitted and with indicated volumes of extracts. Protein concen- added and dTMP incorporation was measured after incubation for tration of the extract of dnaZ ts cells was 27.7 mg/ml; that of incorpo- 20 min at 240. 100% activity for the dnaZ ts extract (-) was 34 dnaZ+ cells was 25.2 mg/ml. After 20 min at 300, dTMP pmol with 4X174 DNA and 14.7 pmol with ST-1 DNA. One hun- ration catalyzed by the following extracts was measured: dnaZ ts dred percent activity for the dnaZ+ extract (0) was 43.5 pmol with (0), dnaZ+ (0), equal mixtures (by volume) of the two extracts OX174 DNA and 13.3 pmol with ST-1 DNA. One hundred percent (0), dnaZ ts supplemented with purified dnaZ gene product (0.05 activity for an equal mixture (by volume) of dnaZ ts and dnaZ+ unit of glycerol gradient fraction) (-), and dnaZ+ supplemented extracts (C) was 67 pmol with 4X174 DNA and 13.1 pmol with with dnaZ gene product (a). ST-1 DNA. One hundred percent activity for the dnaZ ts extract which was supplemented with purified dnaZ gene product (0.05 suggesting that extracts were limiting in a single heat-labile unit of glycerol gradient fraction) during the 240 incubation (M component. Extracts isolated from dnaZ ts cells which was 56.5 pmol with OX174 DNA and 15.2 pmol with ST-1 DNA. showed no fd DNA-dependent DNA synthesis were specifi- cally stimulated by purified dnaZ gene product (Fig. 2). were as above with the omission of rifampicin and the addi- Similarly prepared extracts from dnaZ+ cells showed fd tion of 0.2 mM each of UTP, CTP, and GTP and 300 pmol DNA synthesis which was not further stimulated by the ad- of fd DNA. Reactions with 4X174 and fd DNA were incu- dition of dnaZ gene product. Equal mixtures of dnaZ ts and bated 20 min at 300 and those with ST-1 DNA for 10 min at dnaZ+ extracts showed more than additive activity, again 300. After incubation, trichloroacetic acid-insoluble radioac- suggesting an excess of dnaZ gene product in wild-type ex- tivity was measured. One unit of dnaZ activity stimulated tracts. Thus the dnaZ gene product is clearly required for incorporation of 1 nmol of dTMP under the above condi- conversion of 4PX174, ST-1, and fd DNAs to duplex struc- tions with 4X174 DNA. All three assays were linear with in- tures. creasing amounts of dnaZ gene product over a 5- to 10-fold Purification of dnaZ Gene Product. The dnaZ gene range. The relative dnaZ complementing activity in these product was isolated from extracts of uninfected E. coli three assays varied with the particular preparation of ammo- using kX174, ST-1, and fd in vitro complementation assays. nium sulfate fraction of dnaZ ts cells used, but not with the The properties of each complementation assay were identi- dnaZ gene product used. cal to those described before for crude extracts and for the In Vitro DNA Elongation Reaction. Reaction mixtures reconstituted systems; all three assays required DNA, (0.03 ml) contained 20 mM Tris.HCl (pH 7.5), 10 mM dNTPs, Mg+2, and purified dnaZ gene product. In each MgCl2, 4 mM dithiothreitol, 0.5 mg/ml of bovine serum al- assay the DNA synthesized was nearly full-length linear bumin, 0.3 mM ATP, 0.04 mM each of dATP, dCTP, dGTP, DNA. The 4X174 assay was rifampicin insensitive (3) and and [3H]dTTP (500-1000 cpm/pmol), 2 nmol of 4X174 required only ATP of the NTPs, as we have consistently ob- DNA primed with RNA by E. coli RNA polymerase (14), served (2, 16), but in contrast to the results of others (4, 15). DNA elongation factor I (0.1 unit, 0.05 gg), DNA elongation The fd assay was rifampicin sensitive and required all four factor III (0.1 unit, 0.1 ,jg), DNA polymerase III (0.3 unit, NTPs. The ST-1 assay was rifampicin insensitive, did not re- 0.7 ,g), and dnaZ gene product. After 20 min at 300, acid- quire UTP, CTP, or GTP and was stimulated about 2-fold insoluble radioactivity was measured. In the absence of by ATP. The ratio of OX174 to fd to ST-1 activity remained dnaZ gene product 0.1-1.0 pmol of dTMP were incorporat- constant throughout the purification, which was about ed; the reaction was linear with increasing amounts of dnaZ 5000-fold; the yield was about 10% (Table 1). The isolation gene product over a 20-fold range. of dnaZ gene product from dnaZ ts cells and the demonstra- tion of its thermolability in vitro has not been possible. At RESULTS present there is only one dnaZ ts mutant (AX727) and the Requirement for dnaZ Gene Product in In Vitro DNA dnaZ activity in this strain has been too labile to allow its iso- Synthesizing Systems. Extracts of E. coli dnaZ ts cells lation. showed increased thermolability of both 4X174 and ST-1 Involvement of dnaZ Gene Product in DNA Elonga- DNA-dependent DNA synthesis when compared to extracts tion. The 4X174, fd, and ST-1 DNA synthesizing systems of dnaZ+ cells (Fig. 1A and B). Equal mixtures of dnaZ ts have been reconstituted with purified protein preparations. and dnaZ+ extracts showed more than additive qX174 and The proteins common to these systems are DNA binding ST-1 activity after heating (Fig. 1), suggesting that extracts protein, DNA elongation factors I and II, and DNA poly- of the wild-type strain contained excess dnaZ gene product. merase III. Each of these proteins was assayed in the three Furthermore, addition of purified dnaZ gene product stiLnu- dnaZ complementation assays and DNA elongation factor II lated heat-inactivated dnaZ ts extracts to the control level, preparations were found to contain dnaZ activity. We pre- Biochemistry: Wickner and Hurwitz Proc. Nat. Acad. Sci. USA 73 (1976) 1055 Table 1. Purification of dnaZ gene product Table 2. Requirements for DNA elongation of primed single-stranded DNA Specific activity Ratio of DNA polymerase Fraction (units/mg) OX174 :fd :ST-1 III II Crude extract dTMP incorporated Streptomycin sulfate Additions (pmol/20 min) Ammonium sulfate 9.7 DEAE-cellulose 84 100:100:100 Complete 28.3 37.4 Phosphocellulose-I 330 100:102:100 - DNA elongation factor I 0.3 <0.2 DNA-agarose 1,290 100:115:96 - DNA elongation factor III 0.3 <0.2 Glycerol gradient 1,600 100:105:112 - dnaZ gene product 0.3 0.2 Phosphocellulose-II 12,500 100:113:95 -DNA polymerase 0.2 <0.2 -DNA binding protein 0.7 In this experiment with the DEAE-cellulose fraction, 1 unit of + Supermidine (3 mM) 9.4 7.9 dnaZ activity in the OX174 assay catalyzed incorporation of 0.54 nmol of dTMP with fd DNA and 0.74 nmol with ST-1 DNA. This Reactions were as described in Materials and Methods with 0.36 ratio is defined as 100:100:100. dnaZ gene product was purified as unit of DNA polymerase I11 or 0.14 unit of DNA polymerase II, 0.2 follows from E. coli strain BT1040. It has been purified from strains unit of DNA elongation factor I, 0.1 unit of DNA elongation factor NY73, E4860, and BT1026; the use of dnaE ts cells facilitated the m, and 0.05 unit of dnaZ gene product. Reactions with DNA poly- removal of DNA polymerase ]I. Crude extracts of wild-type cells merase II included 4 sg of DNA binding protein; reactions with contained about one-tenth as many units of dnaZ gene product as DNA polymerase III did not include DNA binding protein. In of DNA polymerase III; glycerol gradient fractions of dnaZ gene similar reactions with 2, 10, and 50 ng of DNA polymerase I in product contained about three times as many units of dnaZ activity place of DNA polymerase HI, 1.7, 9.3, and 58.6 pmol of dTMP as of DNA polymerase III. Crude extract was prepared (8) from 500 were incorporated, respectively, in the absence of added factors; g of E. coli (volume = 520 ml; 35.2 g of protein). Streptomycin 5.0, 13.3, and 71.2 pmol of dTMP were incorporated, respectively, sulfate supernatant and 40% ammonium sulfate precipitate were with added dnaZ gene product and DNA elongation factors I and prepared (8). The precipitate was resuspended in 50 ml of 15% III; and 9.3, 18.1, and 49.7 pmol of dTMP were incorporated, re- (vol/vol) glycerol, 1 mM dithiothreitol, 1 mM EDTA, and 0.02 M spectively, with added DNA binding protein plus dnaZ gene prod- 4 hr. Tris-HCl (pH 7.5) (buffer A) and dialyzed against buffer A uct and DNA elongation factors I and HI. At each level of DNA The sample was diluted with buffer A to an ammonium sulfate polymerase I used, the addition of DNA binding protein (0.4 gg) concentration of 0.02 M (volume = 450 ml; 3.3 g of protein; 32,000 alone inhibited dTMP incorporation 70%. With 4, 20, and 100 ng of units; 100% yield), applied to a 4 X 40 cm DEAE-cellulose column phage T4 DNA polymerase, 2.1, 7.7, and 19.7 pmol of dTMP were equilibrated with buffer A, and eluted with a 4 liter gradient from incorporated, respectively, in the absence of added factors; 2.2, 0 to 0.5 M KCl in buffer A. Fractions eluting between 0.22 and 0.30 10.2, and 27.0 pmol of dTMP were incorporated, respectively, with M KCl were pooled and adjusted to 50% saturation with solid am- added dnaZ gene product and DNA elongation factors I and III; 1.2, monium sulfate. The precipitate was collected by centrifugation; 10.4, and 23.6 pmol of dTMP were incorporated, respectively, with resuspended in 20% glycerol, 1 mM EDTA, 1 mM dithiothreitol, added DNA binding protein, dnaZ gene product, and DNA elonga- and di- and 0.02 M potassium phosphate (pH 6.5) (buffer B); tion factors I and III. alyzed against buffer B 3 hr. The sample was diluted with buffer B to an ammonium sulfate concentration of 0.02 M (volume = 125 ml; 250 mg of protein; 21,000 units; 66% yield), applied to a 4 x 32 present in DNA elongation factor II preparations was re- cm column of phosphocellulose equilibrated with buffer B and quired. This protein has been isolated and will be referred to eluted with a 3 liter gradient from 0 to 1.0 M NaCl in buffer B. as DNA elongation factor III. Table 2 shows the requirement M were Fractions eluting between 0.23 and 0.30 NaCl concentrated for dnaZ gene product, DNA elongation factors I and III, by ammonium sulfate precipitation as above. The precipitate was suspended in buffer A, dialyzed 3 hr against buffer A, diluted to an and DNA polymerase III or II for elongation of RNA- ammonium sulfate concentration of 0.02 M (volume = 15 ml; 30 primed OX174 DNA. The reaction catalyzed by DNA poly- mg of protein; 10,000 units; 31% yield), and applied to a 1.5 X 10 merase II required in, addition, DNA binding protein. In cm column ofDNA-agarose (8). The column was washed with 45 ml contrast to W. Wickner and Kornberg (14, 22), who reported of buffer A followed by 45 ml of buffer A containing 1 M NaCl. The an absolute requirement for spermidine in DNA elongation, 1 M NaCl eluate was concentrated by ammonium sulfate precipita- we between tion as above (volume = 0.8 ml; 4.5 mg of protein; 5800 units; 18% have had variable results 2-fold inhibition and yield). This fraction was dialyzed against buffer A containing 10% 2-fold stimulation. glycerol and 1 M KCl; 0.2 ml portions were applied to 5 ml of 15- Four lines of evidence suggest the same protein functions 35% glycerol gradients in buffer A containing 1 M KC1. Gradients in the OX174, fd, and ST-1 complementation assays and in were centrifuged for 28 hr at 50,000 rpm in the Spinco SW 50.1 the DNA elongation reaction with purified proteins. (a) = rotor. Active fractions (volume 3 ml; 2.7 mg; 4300 units; 13% dnaZ activity measured in the three complementation assays yield) were dialyzed 2 hr against 20% glycerol, 1 mM EDTA, 1 mM dithiothreitol, and 0.02 M potassium phosphate (pH 7.0) (buffer copurified with constant ratio (Table 1). Furthermore, all C) and applied to a 16 X 1.2 cm phosphocellulose column equil- four activities cosedimented through glycerol gradients (Fig. ibrated with buffer C. The column was eluted with a 200 ml gradi- SA), and coeluted on phosphocellulose column chromatogra- ent from 0.05 to 0.5 M potassium phosphate (pH 7.0) in buffer C. phy (Fig. SB). (b) None of the four activities was present in Active fractions were concentrated by vacuum dialysis against buf- protein fractions prepared from dnaZ ts cells. (c) Stimula- fer C containing 0.5 M KCl (volume = 0.5 ml; 0.15 mg; 1900 units; 6% yield). As shown in Fig. 3B, multiple peaks have been seen on tion in all four assays was completely N-ethylmaleimide sen- phosphocellulose column chromatography. sitive using the procedure described (9). (d) dnaZ activity in all four assays was inactivated about 50% by heating 10 min at 420 and 100% by heating 2 min at 1000. viously defined DNA elongation factor II as a protein re- Characterization of dnaZ Gene Product. dnaZ gene quired with DNA elongation factor I and DNA polymerase product, DNA elongation factor III, and DNA polymerase III or II for elongation of primed single-stranded DNA (11). III could be separated by standard techniques (Fig. 3A). In an attempt to reconstitute the elongation reaction with Furthermore, Ad'Z activity in preparations of dnaE ts cells purified dnaZ gene product, we found that a second protein was no more thermolabile than dnaZ activity in the wild- 1056 'Biochemistry: Wickner and Hurwitz Proc. Nat. Acad. Sci. USA 73 (1976)

40

30 _ 0 20 = a 10a _..a

.0.2

r-"X 0 -0.1 ±J.. i

40 50 FRACTION NUMBER FIG. 3. Copurification of dnaZ activity in various assays. (A) Glycerol gradient sedimentation. A portion of the DNA-agarose fraction was sedimented through a 4.2 ml gradient of 15-35% glycerol containing 1 M KCl and buffer C for 22 hr at 60,000 rpm in the Spinco SW60 rotor. Twenty-eight fractions were collected and assayed for dnaZ gene product with OX174 DNA (0), fd DNA (o), and ST-1 DNA (M) complementation assays and with RNA-DNA hybrid in the elon- gation reaction (A); for DNA polymerase III (-); and for DNA elongation factor III (3). Fraction 13 contained 430 units/ml of dnaZ activity with OX174 DNA. (B) Phosphocellulose-TI column chromatography. dnaZ protein (glycerol gradient fraction) was subjected to phosphocellulose-II chroma- tography as described in Table 1. Fractions were assayed for dnaZ gene product with +X174 DNA (-), fd DNA (0), and ST-1 DNA (A) com- plementation assays; with the RNA-DNA hybrid assay (A); and for protein (v). Fraction 38 contained 105 units/ml of dnaZ gene product with 4X174 DNA. In these two experiments 1 unit of dnaZ catalyzed the incorporation of 1.3, 0.9, and 0.26 nmol of dTMP with fd, ST-1, and RNA-DNA hybrid, respectively. U = unit. type control; DNA polymerase III activity in this fraction found that dnaZ ts cells cease DNA synthesis immediately was inactivated at 10 times the rate of the wild-type control when shifted to the nonpermissive temperature. Our results (Fig. 4A). In addition, dnaZ activity in preparations from show that dnaZ gene product is required for conversion of dnaZ ts cells was not detected; DNA polymerase III activity several single-stranded phage DNAs, including OX174 and was heat inactivated at the same rate as the wild-type con- fd, to duplex DNA in vitro. This is consistent with in vivo trol (Fig. 4B). These results suggest that dnaZ gene product results of Truitt and Walker (1) and Taketo (24), who have and DNA polymerase III can exist separately. found that kX174 and M13 (fd) phage growth requires dnaZ The dnaZ gene product has a native molecular weight of gene product; these phages do not grow at the nonper- about 125,000 as determined by glycerol gradient centrifu- missive temperature in dnaZ ts cells. gation (as described in Fig. 3A) with aldolase (158,000 dal- Our results also show that the dnaZ gene product is in- tons) and malate dehydrogenase (70,000 daltons) as internal volved in the elongation of DNA in vitro catalyzed by DNA markers. Purified dnaZ gene product (glycerol gradient polymerase II or III in combination with DNA elongation fraction) was assayed for several activities. It contained no factors I and III, and that these factors can be separated detectable dnaB, C(D), or G gene products, DNA polymer- from one another. This reaction appears much more com- ase, DNA elongation factors I or III, or DNA binding protein plex than was originally thought by us (10, 11) or by W. (measured as described in Materials and Methods using 0.5 Wickner and Kornberg (14, 22). They observed that DNA unit of dnaZ gene product). In addition to being free of elongation required two protein fractions, DNA polymerase DNA polymerase, it has no effect on DNA polymerase activ- III* and copolymerase III* (14), and that the reaction could ity on DNase-treated salmon sperm DNA (using 0.5 unit of be catalyzed by a single protein preparation, DNA polymer- dnaZ). It did not catalyze detectable NMP incorporation ase III holoenzyme (13). A plausible explanation of dis- with 4X174 or calf thymus DNA as template (using 0.5 unit crepancies between our results and those of W. Wickner and of dnaZ gene product). Kornberg is that DNA polymerase III* preparations contain DNA polymerase III, dnaZ gene product, and DNA elonga- tion factor III; DNA polymerase III holoenzyme prepara- DISCUSSION tions contain DNA polymerase III, dnaZ gene product, and In vtvo data of Filip et al. (23) suggest dnaZ gene product is DNA elongation factors I and III. Consistent with this, all involved in E. coli chromosome elongation. These workers DNA polymerase III* preparations we have tested contain Biochemistry: Wickner and Hurwitz Proc. Nat. Acad. Sc. USA 73 (1976) 1057 viously reported the isolation by agarose gel filtration of an > 100 5 initiation complex requiring for its formation 4X174 DNA; 60 dnaB and dnaC gene products; DNA binding protein; repli- 40 cation factors X, Y, and Z; ATP; and Mg++ (5, 9, 16). DNA ~20- synthesis by this isolated complex requires dnaZ and dnaG gene products, elongation factors I and III, DNA polymerase III, and dNTPs.

U.s 0 We wish to thank M. Gellert and G. Felsenfeld for their support and N. Nossal for kindly providing T4 DNA polymerase. 5 10 20 1,0 15' 20 25 TIME AT 38- (minutes) TIME AT 42- (minutes) 1. Truitt, C. L. & Walker, J. R. (1974) Biochem. Biophys. Res. FIG. 4. Heat inactivation of DNA polymerase III and dnaZ Commun. 61,1036-1042. gene product in preparations from dnaE ts and dnaZ ts cells. 2. Wickner, R. B., Wright, M., Wickner, S. & Hurwitz, J. (1972) (A) Heat inactivation of preparation of dnaE ts cells. Phospho- Proc. Nat. Acad. Sci. USA 69,3233-3237. cellulose-I fractions were prepared as described in Table 1 from 3. Wickner, W. T., Brutlag, D., Schekman, R. & Kornberg, A. dnaE ts cells (BT1040) and from wild-type cells, heated at 380 for (1972) Proc. Nat. Acad. Sci. USA 69, 965-969. the times indicated, and assayed as described in Materials and 4. Schekman, R., Wickner, W. T., Westergaard, O., Brutlag, D., Methods for DNA polymerase III (0, dnaE ts preparation; 0, Geider, K., Bertch, L. L. & Kornberg, A. (1972) Proc. Nat. wild-type preparation) and for dnaZ complementing activity with Acad. Sci. USA 69,2691-2695. OX174 DNA (A, dnaE ts preparation; A, wild-type preparation). A 5. Wickner, S. & Hurwitz, J. (1975) in DNA Synthesis and Its mixture of equal amounts of dnaE ts and wild-type preparations Regulation, eds. Goulian, M. M., Hanawalt, P. C. & Fox, C. F. was also heated and assayed for DNA polymerase III (o). Prior to heating the dnaE ts preparation contained 130 units/ml of DNA (W. A. Benjamin, Inc., Menlo Park, Calif.), 227-238. polymerase III and 44 units/ml of dnaZ activity; the wild-type 6. Bouche, J.-P., Zechel, K. & Kornberg, A. (1975) J. Biol. Chem. preparation contained 290 units/ml of DNA polymerase III and 15 250,5995-6001. units/ml of dnaZ. 7. Wright, M., Wickner, S. & Hurwitz, J. (1973) Proc. Nat. Acad. (B) Heat inactivation of preparation of dnaZ ts cells. Phospho- Sci. USA 70,3120-3124. cellulose-I fractions from dnaZ ts (AX727) and wild-type cells 8. Wickner, S., Wright, M., Berkower, I. & Hurwitz, J. (1974) in were heated at 420 and assayed for DNA polymerase III (o, dnaZ DNA Replication, ed. Wickner, R. B. (Marcel Dekker, Inc., ts preparation; 0, wild-type preparation). Prior to heating the New York), pp. 195-215. dnaZ ts preparation contained 130 units/ml and the wild-type 152 9. Wickner, S. & Hurwitz, J. (1975) Proc. Nat. Acad. Sci. USA units/ml of DNA polymerase III. The DNA polymerase activity in 72,921-925. the dnaZ ts preparation was >95% N-ethylmaleimide sensitive. 10. Hurwitz, J., Wickner, S. & Wright, M. (1973) Biochem. Bio- phys. Res. Commun. 51, 257-267. 11. & DNA polymerase III, dnaZ gene product, and DNA elonga- Hurwitz, J. Wickner, S. (1974) Proc. Nat. Acad. Sci. USA tion III in amounts. 71, 6-10. factor variable 12. Geider, K. & Kornberg, A. (1974) J. Biol. Chem. 249, 3999- The protein structures of DNA polymerase III and these 4005. other DNA elongation proteins are not well understood yet. 13. Wickner, W. & Kornberg, A. (1974) J. Biol. Chem. 249, Otto et al. (25) suggested that DNA polymerase III consisted 6244-6249. of a single subunit of 140,000 daltons. Livingston et al. (19) 14. Wickner, W., Schekman, R., Geider, K. & Kornberg, A. (1973) found two protein species, one of 140,000 and one of 40,000 Proc. Nat. Acad. Sci. USA 70, 1764-1767. daltons, when they subjected to sodium dodecyl sulfate gel 15. Schekman, R., Weiner, A. & Kornberg, A. (1974) Science 186, electrophoresis the single protein species seen on native 987-993. polyacrylamide gel electrophoresis which migrated with 16. Wickner, S. & Hurwitz, J. (1974) Proc. Nat. Acad. Sci USA DNA polymerase III activity. If these proteins were both 71,4120-4124. 17. Gefter, M. L., Hirota, Y., Kornberg, T., Wechsler, J. & Bar- DNA III gene subunits of polymerase then another besides noux, C. (1971) Proc. Nat. Acad. Sci. USA 68,3150-3153. dnaE could influence DNA polymerase III activity. W. 18. Sigal, N., Delius, H., Kornberg, T., Gefter, M. & Alberts, B. Wickner et al. (14) claimed homogeneity of DNA polymer- (1972) Proc. Nat. Acad. Sci. USA 69,3537-3541. ase III* based on sodium dodecyl sulfate gels without corre- 19. Livingston, D. M., Hinkle, D. C. & Richardson, C. C. (1975) J. lating activity and protein. They proposed that DNA poly- Biol. Chem. 250,461-469. merase III was a dimer and DNA polymerase III* was a tet- 20. Wickner, R. B., Ginsberg, B., Berkower, I. & Hurwitz, J. ramer of identical 90,000 dalton subunits, each coded by the (1972) J. Biol. Chem. 247, 489-497. dnaE gene. To date, we have no data on the subunit struc- 21. Jovin, T., Englund, P. & Bertsch, L. (1969) J. Biol. Chem. 244, ture of DNA polymerase III or the other DNA elongation 2996-3008. factors. 22. Wickner, W. & Kornberg, A. (1973) Proc. Nat. Acad. Sci. USA 70,3679-3683. Thus, the dnaZ gene product functions in the elongation 23. Filip, C. C., Allen, J. S., Gustafson, R. A., Allen, R. G. & Walk- of DNA strands with DNA elongation factors I and III and er, J. R. (1974) J. Bacteriol. 119,443-449. DNA polymerase III or II. In the conversion of kX174 sin- 24. Taketo, A. (1975) Mol. Gen. Cenet. 139,285-291. gle-stranded DNA to duplex DNA, the dnaZ gene product 25. Otto, B., Bonhoeffer, F. & Schaller, H. (1973) Eur. J. Bio- functions in the elongation phase of the reaction. We pre- chem. 34, 440-447.