Eur. J . Biochem. 201, 147-155 (1991) ‘(3 FbBS 1991 001429569100615Q

The polymerase domain of Streptococcus pneumoniae DNA polymerase I High expression, purification and characterization Maria Elena PONS’, Asuncion DiAZ

‘, Sanford A. LACKS’

and Paloma LOPEZ’

Centro de Investigaciones Biologicas, Madrid, Spain Brookhaven National Laboratory Upton, USA (Received April 24, 1991)

~

EJB 91 0532

The 3‘-terminal two-thirds of the Streptococcus pneumoniae polA gene was cloned in an Escherichia coli genefusion vector with inducible expression. The resulting recombinant plasmid (pSM10) directs the hyperproduction of a polypeptide of 70.6 kDa corresponding to the C-terminal fragment of pneumococcal DNA polymerase I. Induced cells synthesized catalytically active protein to the extent of 7% of the total soluble protein in the cells. The polymerase fragment was purified to greater than 90% homogeneity with a yield of 1.5 mg pure protein/ culture. The protein has DNA polymerase activity, but no exonuclease activity. The enzyme requires a divalent cation (MgC1, or MnCl,) for polymerization of DNA. Comparison of the mutant and wild-type pneumococcal polymerases shows that the construction did not affect the enzymatic affinity for the various substrates. The mutant protein, like its parent DNA polymerase 1, exhibited an intermediate level of activity with primed single-stranded DNA. At high molar ratio of enzyme/DNA Substrate, the polymerase fragment catalyzes strand displacement and switching after completing the replication of a primed single-stranded M 13 DNA molecule.

DNA polymerase I of Escherichia coli is involved in DNA repair and in the processing of Okazaki fragments during replication of DNA [I]. The enzyme has three enzymatic activities contained in a single polypeptide, 5’ + 3’ and 3’ + 5’ exonucleases and DNA polymerase [ 11. These activities were ascribed to different domains of the protein by controlled proteolytic digestion [2] and by cloning of DNA encoding either the Klenow fragment [3] or the polymerase domain [4]. The polA gene of Streptococcus pneumoniae has been cloned [5] and sequenced [6] and purified its product. In addition, the entire gene has been placed downstream of the 410 T7 promoter and the pneumococcal DNA polymerase 1 has been hyperproduced in E. coli under the control of the T7 RNA polymerase [5]. This protein, the pneumococcal counterpart of E. coli DNA polymerase I, possesses exonuclease and polymerase activities [6]. Comparison of the protein sequences deduced from the DNA sequences of both bacterial DNA polymerases showed an overall amino acid identity of 40%. Since the E. coli Klenow fragment was crystallized [7], it was possible to compare the three-dimensional structure of the Klenow fragment to the pneumococcal DNA polymerase 1. A high degree of similarity was detected within the polymerase domain of the Klenow fragment [6], particularly in the cleft that binds duplex DNA [7, 81. However, the pneumococcal Correspondence to P. Lopez, Centro de Investigaciones Biologicas, Velazquez 144, E-28006 Madrid, Spain Abbreviation. IPTG, isopropyl thio-/I-D-galactoside. Enzymes. C-terminal fragment of S. pneunzoniae DNA polymerase I, E. coli DNA polymerase I, E. coli Klenow fragment and S. pneumoniue DNA polymerase I (EC 2.7.7.7); type-I1 restriction endonucleases AvaII, BamHI, BglII and EcoRI (EC 3.1.21.4); T7 lysozyme (EC 3.2.1.17). Note. The novel nucleotide sequence data published here h a w been submitted to the EMBL sequence data bank.

enzyme differed considerably from that of E. coli in the putative domain of the 3’ 45’ exonuclease activity. In addition, the S. pneumoniae DNA polymerase I is 51 residues smaller than that of E. cofi. To further characterize the pneumococcal DNA polymerase I, the construction of a plasmid was attempted that would express a polypeptide equivalent to the E. coli Klenow fragment. It was found that the C-terminal fragment of the S. pneumoniae DNA polymerase I (residues 269- 877) contains the domain for the polymerase activity, but does not show any delectable exonucleolytic activity. The protein has been purified, characterized and compared to the Klenow fragment and to the pneumococcal DNA polymerase I. MATERIALS AND METHODS Bacterial strains and plasmids

The strains used were E. coli BL21(DE3) (ompT hsdS gul F- [int::P,,,,,,. T7 gene I imm21 nin.51) [9], and S. pneumoniue 708 (end-I exo-2 trt-1 hex4 malM.594). Plasmids used were pSM22 [5], pLysS [9], PET-3b [lo], pLSl [ l l ] and pSMl0 (described in this work). Purified plasmids were prepared from E. coli BL21(DE3) [pET-3b], E. coli BL21(DE3) [pLysS] [pSM10] and S. pneumoniae 708 [pSM22] according to Currier and Nester [12]. Cloning procedure Our objective was to clone a DNA fragment containing the coding sequence for the putative 3’ + 5’ exonuclease and polymerase domains of S. pneumoniar DNA polymerase I. Plasmid pSM22 (Fig. l A , B) was digested with AvaII and EcoRI, and a 1.8-kb AvaII - EcoRI DNA fragment containing

148 Hind m

A

e R !

pSM22 Ava P

I AvaU

B

BamHI L

EcoRI I

EcoRI

C

D Fig. 1. Construction ofpSM10 and itsfusion junction. (A) Physical maps of PET-3b and pSM22. Arrows indicate the direction of transcription of the genes. In pSM22 line and box segments indicate pLSl vector plasmid and pneumococcal chromosomal insert, respectively; shaded segment, pneumococcal polA gene. In pET-3b, the location of the T7 promoter ($10) and of the translation-start site (s10) for gene 10 are indicated. (B) DNA fragments used for construction of pSMl0. (C) Physical map of pSM10. (D) DNA nucleotide sequence from pSMlO downstream of the gene 10 initiation codon and the 24 first amino acids predicted for the fused protein. The arrowhead indicates the junction point generated in the construction of pSM10. Determination of the N-terminal sequence of the purified C-terminal fragment of the pneumococcal DNA polymerase I revealed the residues marked by asterisks

the 3'-terminal two-thirds of the S. pneumoniae polA gene was separated by electrophoresis on agarose gel and purified by binding and elution from glass powder (Gene Clean kit, BIO 101, Inc.). Plasmid PET-3b (Fig. l A , B) was digested with BamHI and EcoRI and a 4.1-kb BamHI - EcoRI DNA fragment was purified as above. The 5' EcoRI overhangs from the 1.8-kb and 4.1-kb DNA fragments were ligated together. Afterwards, the 5' overhangs made by AvaII and BamHI treatment were filled in with the E. coli Klenow fragment and the blunt ends were ligated. This DNA was transformed into E. coli BL21(DE3) [pLysS] by the method of Kushner [I31 and ampicillin-resistant clones were identified. Plasmid contents of 24 transformants were screened by the alkaline lysis procedure [I41 and analyzed by agarose gel electrophoresis; five contained a 5.9-kb recombinant plasmid and one of these was designated pSMlO (Fig. 1 C). Cloning the DNA containing the fragment of the polA gene into the BamHI site of PET-3b should produce an in-frame fusion after the first 11 amino acids of the gene 10 protein [lo].

Determination of the 250 nucleotides of the DNA sequence of pSMlO starting from its BglrI site, by the chemical method of Maxam and Gilbert [15], showed that the fusion junctions present in pSMlO were as expected (Fig. 1 D). No rearrangements occurred in the transcription and translation signals for T7 gene 10. Analysis ?f plasmid-encoded proteins The E. coli BL21(DE3) host-vector cloning system [9] allows the specific radiolabeling of protein products encoded by genes in the plasmid [16]. Details of the procedures used for radiolabeling and analyzing proteins have been described previously [ll]. Protein analysis Protein concentration was determined by measurement of absorbance at 280 nm and corrected for absorbance at

149 Table 1. Analysis of purification scheme The yield of polymerase activity in crude extracts, 74700 U was set as 100% Sample

Crude extract Streptomycin sulfate supernatant Ammonium sulfate extract Bio-Gel A-0.5m fractions 44-49 Hcparin-agarose fractions 23 -29 DEAE-Sephacel fractions 27 - 29

Fig. 2. Steps oi purification of the C-terminalfragment of DNA polymerase Z ( P o l l * ) . (A) 12% SDSjPACE stained with Coomassie blue (lanes 1 -6) and autoradiogram of dried gel previously developed for polymerase activity (lanes 7-9). Lane 1 , standard protein (top to bottom: rabbit phosphorylase, bovine serum albumin, chicken ovalbumin, bovine carbonic anhydrase and soybean trypsin inhibitor); lane 2, crude extract; lanes 3 and 7, ammonium sulfate precipitate; lane 4, peak of gel-filtration column; lane 5 , peak of heparinagarose column; lanes 6 and 9, peak of DEAE-Sephacel column; lane 8, purified E. coli DNA polymerase I (Eco PolI). Appropriate volumes of each sample were applied to the gel to give 6 pg/well (lanes 2 -4) or 3 pg/well protein (lanes 5 - 6) or 3 U/well polymerase activity (lanes 7-9). (B) 0.8% agarose gel stained with ethidium bromide. 0.2 pg covalently closed (BI) or linearized (B2) pLSl plasmid was incubated for 4 h (lanes 1 -4) or 20 h (lanes 6-9) with 2 pI peak of the gelfiltration column (lanes 2 and 7); 2 p1 peak of the heparin-agarose column (lanes 3 and 8), 2 pl peak of the DEAE-Sephacel column (lanes 4 and 9). Lanes 1 and 6, DNA control; lanc 5, molecular mass standards, phage T7 DNA digested with MboI

260 nm. The proportion of the C-terminal fragment of the DNA polymerase I in crude extracts was performed by soft laser densitometric scanning of the gels in a LKB Ultroscan 2202 coupled to an Apple 11 computer. SDSjPAGE was carried out as in [17]. Gels were stained with Coomassie brilliant blue R260. Protein purijication A 500-ml culture of E. coli BL21(DE3) [pLysS] [pSMlO] was grown in M9 medium supplemented with 200 pg/ml ampicillin at 37°C to A,,, of 0.5; at this time, isopropyl-thiofl-D-galactoside (IPTG) at 0.5 mM was added, and the culture was incubated for 3 h more. Cells were concentrated 20-fold in buffer A (20 mM TrisjHCl, pH 7.6, 1 mM dithiothreitol, 1 mM EDTA). Crude extract was prepared by three cycles of freezing and thawing at - 70°C and 37"C, respectively. The presence of T7 lysozyme encoded by the plasmid pLysS allows efficient lysis of the cells in this procedure. Removal of cell debris was accomplished by centrifugation. The supernatant fluid was designated as the crude extract (Table 1; Fig. 2A, lane 2). Streptomycin sulfate was added to the crude extract to a final concentration of 5.8%. The suspension was allowed to stand for 30 min and was centrifuged. Ammonium sulfate to 65% saturation was added to the supernatant fluid (designated as streptomycin sulfate supernatant). The resulting pre-

Protein

Specific activity

Yield of activity

mg

U/mg

Yo

87.45

854

83.82 55.35

694 1250

77.9 92.8

9.81

5200

68.3

2.03

13970

38.0

0.73

27070

36.2

100

cipitate was dissolved in buffer B (buffer A supplemented with 50 mM NaCl and 5% ethylene glycol) and was designated as ammonium sulfate extract (Table 1, Fig. 2A, lane 3). This material was applied to an agarose column (Bio-Rad Bio-Gel A-OSm, 85 c m x 1.6 cm) and eluted with buffer B. A major peak of polymerase activity was detected in fractions 42- 53. Analysis of this peak by SDSjPAGE revealed the presence of several protein bands (Fig. 2A, lane 4) and the polymerase activity was ascribed to two proteins by polymerase assay in the gel (Fig. 3A, inset): the expected 70.6-kDa polypeptide, and a protein with a mobility in SDSjPAGE corresponding to the E. coli DNA polymerase I (103 kDa). Fractions 4350, were subsequently fractionated on a 10 ml (Bio-Rad, 15 cm x 0.9 cm) heparin-agarose column equilibrated with buffer B. Bound proteins were eluted at 10 mljh with a 100ml linear gradient (50-SO0 mM) NaCl in buffer B. The pneumococcal polymerase eluted as a peak at fractions 21 31, as detected by polymerase activity (Fig. 3 B, inset) and by SDSjPAGE (Fig. 2A, lane 5). Fractions 22-29 from the heparin-agarose column were pooled and diluted twofold with buffer B without NaCl. This sample was applied to a 10-ml DEAE-Sephacel column (Pharmacia, 15 cm x 0.9 cm). The column was developed at 12 ml/h with a linear gradient (50 500 mM) NaCl in buffer B. The C-terminal fragment of S. pneumoniae DNA polymerase 1 eluted at 0.21 M NaCl (Figs 3 C and 2A, lanes 6 and 9). Fractions containing polymerase activity were pooled and stored at -20°C after adding an equal volume of glycerol. No loss of polymerase activity was detected in the protein preparation after 6 months of storage at - 20 "C. Enzyme assays Polymerase activity was determined by incubating samples for 15 min at 37°C in 50 p1 of a mixture containing 45 mM Tris/HCl, pH 8, 1.25 mM 2-mercaptoethanol, 6.5 mM MgC12, 100 mM KC1, 45 pg bovine serum albumin, 1 pg salmon sperm DNA (previously nicked with pancreatic deoxyribonuclease I), SO pM each of dATP, dGTP and dCTP, 15 pM dTTP, and 100 nCi [3H]dTTP at 40 Ci/mrnol. Reactions were terminated by chilling to 0 "C, and [3H]dTTPincorporated into DNA was measured as previously described [6]. In kinetics studies, dATP, dGTP and dCTP were omitted and salmon sperm DNA was substituted by poly(dA). (dT),, at the concentrations indicated in Results. In these experiments,

150 [a-32P]dCTPand all four unlabeled deoxynucleoside triphosphates. 3’ -+ 5’ exonuclease activity was determined in samples containing 50 mM Tris/HCl (pH 7.6), 2 mM MnC12, 3 mM 2mercaptoethanol and 25 ng heat-denatured j2P-labeled DNA. After incubation at 37°C for the times incubated in Results, release of acid-soluble 32P from the end-labeled DNA was measured as previously described [6]. Primer-extension assays

>-

--

I-

8

-

6

L

4 2

25

26 27 28 29 30 31

i5b a -

:I in

2

5

10

15

20

25

30

35 40

FRACTIONS Fig. 3. Purification ojthe C-terminal fragment of DNA polymeruse I. (A) Agarose gel filtration. A 65% ammonium sulfate fraction was applied to a column of Bio-Gel A-0.Sm an eluted. (B) Fractionation on heparin-agarose column of fractions 44-49 from (A). (C) Fractionation on DEAE-Sephacel column of fractions 23-29 from (B). ( 0 )Polymerase; ( A ) protein concentration; (0)NaCl concentration. The insets are as follows: (A, B) autoradiograms of dried 12% SDS/ PAGE previously developed for polymerase activity; (C) 12% SDS/ PAGE stained with Coomassie blue. 3 p1 fractions 44-52 from (A), 7 pl fractions 21 -30 from (B) and 10 ~1 fractions 25-31 from (C) were analyzed. In insets, arrows indicate position of the C-terminal fragment of DNA polymerase I

0.04 U of either DNA polymerase I or its C-terminal fragment were added to the reaction mixture and samples were incubated for 5 min at 37°C. In these conditions, less than 10% of the substrates were used. 1 U polymerase activity is defined as the amount of enzyme catalyzing the incorporation of 10 nmol dNTP/30 min at 37%. Detection of DNA polymerase in gels was accomplished by a method for revealing enzyme activity after SDSjPAGE [18], as modified for this enzyme [19]. To test for 3’ + 5’ exonuclease activity, a 2-kb NcoI DNA fragment was 32Plabeled at its 3’ ends by filling in the overlap with the E. coli Klenow fragment in the presence of

MI 3mp2 single-stranded DNA was prepared as a template for DNA polymerase assay as described by Kunkel [20]. 5‘ 32P-labeled 5’-d(GTAAAACGACGGCCAGT)-3‘ was prepared and annealed to M13mp2 single-stranded DNA as described by Tabor et al. [21]. For the primer-extension assays, the reaction mixture (18 pl) contained 45 mM Tris/HCl, pH 8, 6.5 mM MgC12, 3 mM 2-mercaptoethanol, 0.3 mM dGTP, dATP, dCTP and dTTP, and l o n g primer template. The reaction mixture was incubated at 37°C for 1 min, then the reaction initiated by the addition of the enzyme (2 pl of a dilution in 45 mM Tris/HCl, pH 8, 5 mM 2-mercaptoethanol and 0.5 mg/ml bovine serum albumin). Reactions were stopped by the addition of 2 p10.5 M EDTA. For analysis by denaturing PAGE, 3 1.11 reaction mixture was added to 3 p1 80% formamide, 10mM NaOH, 1 mM EDTA, 0.1% bromophenol blue and 0.1% xylene cyanol, heated at 95°C for 2 min, and loaded onto an 8 % polyacrylamide gel containing 7 M urea. For analysis by neutral agarose gel electrophoresis, 5 p1 reaction mixture, together with 5 pl 50% glycerol, 0.2% SDS, 25 mM EDTA and 0.1% bromophenol blue, were loaded onto a 0.8% gel containing ethidium bromide at 0.5 pg/ml. Electrophoresis was carried out for approximately 14 h at 12 mA. For analysis by alkaline agarose gel electrophoresis, 5-pl samples were diluted with 5 pl 100mM NaOH, 1 0 m M EDTA, 6% Ficoll, 0.025% bromocresol green and 0.5% xylene cyanol, the loaded onto a 0.8% alkaline agarose gel prepared as described by Sambrook et al. [22]. Electrophoresis was carried out at 50 mA for 16 h. Gels were dried then autoradiographed on Kodak X-Omat S films. N-terminal protein sequence determination

A sample of the electrophoretically pure C-terminal fragment of DNA polymerase I was dialyzed against NH4HC03, 5 mM dithiothreitol and 0.01% SDS. The sample was lyophilized and dissolved in 0.4 ml water four times to eliminate NH4HC03. Approximately 3 nmol was subjected to Nterminal sequence determination in a gas-phase sequencer (Applied Biosystems).

RESULTS AND DISCUSSION Expression of the C-terminal fragment of D N A polymerase I

The suitability of E. coli BL21(DE3) [pLysS] [pSM10] as a source of the C-terminal fragment of DNA polymerase I was tested by induction of the cells with IPTG and pulse labeling with [35S]methionine. Whole cell extracts were analyzed by SDSjPAGE and staining with Coomassie blue (Fig. 4A), and by autoradiography of the dried gel (Fig. 4B). One strong band was visible at 71 kDa for the E. coli strain

151 Table 2. Requirements of the polymerase uctivity qf the C-terminal frugmenl of ihe D N A polymerase 1 Polymerase activity is expressed as the percentage of activity detected in the assay conditions described in Materials and Methods (6.5 mM MgC12, 100 mM KCI) using 0.02 U purified enzyme. BSA, bovine serum albumin System

Activity

Yo Complete -

MgC12

+ 1 mM MgC12 + 3 mM MgClz Fig. 4. Hyperexpression qfthe C-terminalfrugment q f D N A polymerase 1 in E. coli. (A) Gel stained for proteins with Coomassie brilliant blue. (B) Autoradiogram of the dried gel. Lanes 1-4: extracts of E. coli BL21(DE3)[pSM10]; lanes 5 - 8 , extractsofE. coliBL21(DE3) [PET3 b]; lane 9, standard proteins (as in Fig. 2). Extracts wcrc prepared from cells incubated with IPTG as follow: lanes 1 and 5 , 3 h ; lanes 2 and 6, 2 h; lanes 3 and 7, 1 h; lanes 4 and 8, 30 min. After pulselabeling with [35S]methionine for 5 min, extracts were subjected to 5 - 25% SDS/PAGE. Arrow, C-terminal fragment of DNA polymerase I

harboring pSMlO (Fig. 4, lanes 1-4). This band was not detected in the E. coli strain carrying only the plasmid vector PET-3b (Fig. 4, lanes 5-8). The molecular mass of the protein, 71 kDa, corresponded to that expected from the open reading frame cloned in pSM10,70.6 kDa. The amount of the protein and its rate of synthesis increased with the time of induction (Fig. 4, lanes 4- 1). Densitometric scanning of the protein gel stained with Coomassie blue showed that the proportion of the hyperexpressed polypeptide to the total cellular proteins increased to 3% at 1 h (Fig. 4A, lane 3) and 12% at 3 h of induction (Fig. 4A, lane 1). The soluble component corresponded to about 7% of soluble cellular proteins present in the crude extract used for purification of the enzyme (Fig. 2A, lane 2). After 3 h of induction, E. coli BL21(DE3) [pLysS] [pSM10] showed a level of polymerase activity of 1.3 U/pg, about 100 times higher than that detected in E. coli BL21(DE3) [pLysS] [pET-3b]. This difference can be ascribed to the hyperproduced 70.6-kDa protein. Purlficution of the C-terminu1,fragrnent o j D N A polymeruse I

Cells containing pLysS and pSMl0 were induced for 3 h with IPTG and lysed. The homogeneous C-terminal fragment of DNA polymerase I was obtained as described in Materials and Methods. Briefly, this entailed removal of nucleic acids by streptomycin sulfate precipitation, selective protein precipitation with ammonium sulfate and three chromatographic steps of purification. Successive fractionation by gel filtration, heparin-agarose, and DEAE-Sephacel chromatography, yielded a purified enzyme corresponding to more than 90% of the protein present. A summary of the purification is presented in Table 1 and Fig. 2A. It yielded 0.73 mg protein with a specific activity of 27 U/pg from 500 ml culture. This recovery corresponds to 36% of the polymerase activity detected in crude extracts (Table 1). The purified preparation of the C-terminal fragment of S. pneumoniue DNA polymerase I showed less than 10'/0

+ 10 mM MgClz + 20 mM MgC12 + 0.03 mM MnClz + 0.1 mM MnCI2 + 0.3 mM MnCI, + 1.O mM MnCI, + 6.5 mM CaClz

+ 6.5 mM ZnCll

100 0.2 63 92 93 82 52 90 56 25 0.4 0.3

-

KCI

43 91 62 18 3 0.15

-

BSA 2-Mercaptoethanol

37 99

+ 65 mM KCI + 200 mM KCI + 65 mM NaCl + 100 mM NaCl + 200 mM NaCl -

impurities, as measured by Coomassie blue stain (Fig. 2A, lane 6). The preparation is free of contamination by host polymerases (Fig. 2A, lane 9). The E. coli DNA polymerase I detected in the ammonium sulfate extract (Fig. 2A, lane 7) and in the gel-filtration peak (Fig. 3A, inset) was eliminated after fractionation on the heparin-agarose column (Fig. 3 B, inset). To test for the presence of nucleases, linearized and covalently closed circular plasmid DNA were incubated with the enzyme preparation at the different steps of purification for 4 h and 20 h at 37 "C and analyzed in agarose gels (Fig. 2 B). Endonuclease (Fig. 2B1, lanes 2 and 7) and possible exonuclease (Fig. 2B2, lanes 2 and 7) activities were detected in the gel-filtration peak. Apparent exonuclease activities were eliminated after fractionation by heparin-agarose (Fig. 2 B2, lanes 3 and 8). The endonuclease activities still present in the heparin-agarose peak (Fig. 2B1, lanes 3 and 8) were eliminated after DEAE-Sephacel fractionation; neither endonuclease or exonuclease activities wree detected after this step (Fig. 2B, lanes 4 and 9). Properties of the purified C-terminal fragment of D N A polymeruse I The purified protein migrated at the position on polyacrylamide gels expected for 70.6 kDa (Fig. 2A). N-terminal amino acid sequence analysis showed the protein to correspond to that predicted from the DNA sequence; 21 out of 23 residues were accurately identified (Fig. 1 D). The yield of amino acid derivatives was only 15% of that expected, probably due to formylation of the N-terminus in the majoritp of protein molecules. In addition, the initial methionine residue was not detected, presumably due to removal of N-formylmethionine from a fraction of the protein product after translation.

152 Table 3. Polymerase activity of the pneumococcal D N A polymerase I and of its C-terminal fragment on diffrrent substrates Assay conditions were as described in Materials and Methods except that KC1 was omitted in some cases. DNA concentration was 120 pM for poly(dA) . (dT),,, 20 pg/ml for activated salmon sperm DNA and 50 pg/ml for primed single-stranded M13mp2 DNA [KCI]

Substrate

Relative polymerase activity intact Poll enzyme

mM

C-terminal fragment

U -

poly(dA). (dT),o

0 100

10600 16200

29 400 20 600

Activated salmon sperm DNA

0 100

6700 16700

10 600 27 500

Primed single-stranded DNA

0 100

1800 3000

6200 2600

0

I0 20 39 I/[TTP] (rnM-’)

Fig. 6. Products ofsequencing reactions using the C-terminul fragment of’DNApolymerase I. DNA sequences of M13mp8 (A) and M13mp2 (B) were determined by the dideoxy-chain-termination method using the MI3 sequencing kit (Amersham) and the C-terminal fragment of the pneumococcal DNA polymerase 1 (A-T) or the E. coli Klenow fragment (A’-T’). (A) M13mp8 was primed with the heptadecameric universal primer and DNA was labeled with [ U - ~ ~ P I ~ A (B) TP, MI 3mp2 was primed with 5’-d(GGGCCTCTTCGCTATTA)-3’, and DNA was labeled with 35S-labeled deoxyadenosine 5’-[a-thio]triphosphate. The assay conditions were as recommended by the supplier for the E. coli Klenow fragment. Markers refer to the number of nucleotides incorporated. Arrow, position of the spurious band. Indicated temperatures refer to the polymerization reaction

DNA after 2 h in the presence of 5 U of the enzyme, whereas with only 0.1 U of the E. coli Klenow fragment 41% of the 2 0 40 60 80 100 radioactivity was acid soluble after a 10-min incubation (data I / [ poly (dA).(dT)lo](mM-’) not shown). The lack of 3‘ --f 5’ exonuclease activity in the pneumococcal enzyme could be due either to improper folding of the corresponding domain in the fused protein or to the lack of the 3’ 5‘ exonuclease domain in the construction. The 5‘ + 3‘ exonuclease activity of the S. pneumoniae polymerase was also tested: less than 5% radioactivity was hydrolyzed from 5’-end-labelled DNA after 2 h in the presence of 5 U of the enzyme (results not shown). --f

Characterization of’ the polymercue activity 5

10

15

20

25

I / [ T T P ] (mM-’)

10

20

30 40

50

I/[poly (dA).(dT),&nM-’)

Fig. 5. K, determination for d T T P a n d p o l y ( d A ) . (LIT),,utilization by Poll ( A ) and ( B ) andits C-termina1,frugment ( C ) and ( 0 ) . (A, C) l / V plotted as a function of l/[dTTP] concentrations at the indicated fixed concentrations of I/[poly(dA) . (dT)lo]. (B, D) l/Vplotted as a function of l/[poly(dA) . (dT),,] concentration at the indicated fixed concentrations of dTTP. Each measurement was carried out in triplicate. The double-reciprocal plot was fitted by a least-squares regression analysis

On the basis of the homology between S. pneumoniae and E. coli DNA polymerases I [6],the construction of plasmid pSMl0 was initially designed to obtain a pneumococcal gene product similar to the E. coli Klenow fragment. However, although the purified protein had polymerase activity, it did not show exonuclease activity. When we tested the 3 ‘ 4 5 ‘ exonuclease activity of the S. pneumoniae polymerase, less than 4% radioactivity was hydrolyzed from 3’-end-labeled

The purified product was used to establish optimal conditions for DNA polymerization with activated salmon sperm DNA (Table 2). The highest values of polymerase activity were obtained in the presence of 6.5 mM MgC12 and 100 mM KCl. When the divalent cation was omitted from the reaction mixture, the polymerase activity was reduced to 0.2%. 90% of the polymerase activity was detected when MgCI2 was substituted by 0.1 mM MnC12. No significant activity was detected in the presence of CaCI2 or ZnC1, instead of MgC12. These results show that the pneumococcal DNA polymerase I fragment requires either Mg2+or Mn2+divalent cations for its polymerizing activity. Removal of KCl reduced the activity to 43%, and its substitution by NaCl decreased the activity even further. When bovine serum albumin was completely omitted from the reaction, the polymerase activity was reduced to 37% of the control (45 pg bovine serum albumin/ assay). Removal of 2-mercaptoethanol had no effect. Table 3 shows a comparison of the polymerizing activity of the pneumococcal DNA polymerase I and its C-terminal fragment with different substrates in the presence and absence of KCl. We also compared the kinetic parameters of the C-

153

A

B

Fig. 7. Processivity of the pneuwlococcal D N A polymerase I (Poll), its C-terminalfragment (Poll*) and E. coli Klenow.frugment ( K l ) . The primer was the 5' 32P-labeled decamer annealed to M 13mp2 single-stranded DNA. The molar ratio of polymerase/primer template was 1 : 100, 1 : 10, 1 : 1 or 10: 1 as indicated. The specific activity of the enzymes were 10.6 U/pg for the pneumococcal DNA polymerase I, 29.4 U/pg for the C-terminal fragment of DNA polymerase I and 6.0 U/pg for the E. coli Klenow fragment. Primer-extension reactions were performed as described in Materials and Methods in the absence (A) or presence (B) of 100 mM KCI. At 0 s (c), 20 s, 1 min and 3 min, samples were removed and analyzed in an 8% polyacrylamide sequening gel. Markers refer to the number of nucleotides incorporated. The sequence of the major pause site for the pneumococcal polymerases is indicated

terminal fragment of pneumococcal DNA polymerase I to those of the intact pneumococcal DNA polymerase I. In order to do this, poly(dA) . (dT),,-directed DNA synthesis was analyzed while varying independently the concentration of each of the template primer and dTTP substrates. Fig. 5A and C shows the plots of l / V versus [dTTP]-' at a fixed concentration of the proteins with different concentrations of poly(dA) . (dT),,. A family of intersecting lines was obtained for both pneumococcal enzymes. A similar pattern was observed for the reciprocal case of poly(dA) . (dT)lo and dTTP as the variable and constant substrates, respectively (Fig. 5 B and D). The K, values obtained with the C-terminal fragment of DNA polymerase I were 14 pM and 36 pM (Fig. 5 D and C), and with intact DNA polymerase I, 10 pM and 25 JLM (Fig. 5 B and A) for poly(dA) . (dT),, and dTTP, respectively. A similar affinity, K , 18 pM for dNTP was observed for the E. coli Klenow fragment [4] (unpublished data). These results show that the truncated C-terminal fragment of the S . pneumoniae DNA polymerase I retains the same affinities as its parental protein for the dNTP and DNA substrates. However, the V,,, was different for the two enzymes 58.2 pmol . min-' . mg enzyme-' and 18.8 pmol . min-'

. mg enzyme-' for the intact DNA polymerase I and the Cterminal fragment, respectively. As a consequence, the rate of synthesis is lower for the truncated protein polymerase than for the DNA polymerase I (23 mol nucleotide . s - . mol enzyme-' versus 103 mol nucleotide . s-' . molenzyme-'. This could result from either a longer cycling time or a lower processivity (probability of dissociation) of the truncated enzyme. The fidelity of the protein in the polymerization of DNA was tested by using the purified fragment for DNA sequence analysis by the dideoxy-chain-termination method [23]. The C-terminal fragment of pneumococcal DNA polymerase I required a 1000-fold excess of ddTTP to block dTTP incorporation (data not shown). The results presented in Fig. 6 indicate that the protein discriminates between deoxynucleotides and dideoxynucleotides in the same fashion as the Klenow fragment, and that it is able to correctly incorporate nucleotides into a template DNA. Increasing the temperature from 21 "C to 37 "C in the polymerization reaction did not change the sequencing pattern, but eliminated spurious bands at the position corresponding to a length of 200 nucleotides (Fig. 6A).

154

A

B

in length, which indicates that most of the enzyme molecules were unable to translocate along the DNA template after incorporation of these nucleotides. In the same conditions, the E. coli Klenow fragment dissociated from the template after incorporation of about 10 nucleotides. The processivity observed for the E. coli enzyme agrees with that previously described [21, 241. The presence of KCl in the processivity experiments (Fig. 7 B) impaired the polymerization catalyzed by both the C-terminal fragment of pneumococcal DNA polymerase I and the Klenow fragment. These results presumably resulted from increased dissociation of the enzyme from the DNA substrate at pause sites. At a molar ratio of 1:lO of enzyme/DNA substrate, from 2 s to 3 min of polymerization, only 3 - 14% of the elongated molecules reached the 29 34 nucleotides in length and 60-57% of the polymerized molecules were still present at positions shorter than 10 nucleotides. In contrast, in the same conditions, when KCI Fig. 8. Strund displacement cuiulyzed by the C-terminal fragment of was omitted (Fig. 7A), polymerization of less than 29 D N A polymeruse I. (A) Autoradiogram of alkaline (A) and neutral (B) agarose gels. Reaction conditions are described in legend to Fig. 7. nucleotides occurred only in 30 - 39% of the elongated molecules. The influence of the ionic strength was less pronounced Reactions were perrormed in the absence (lanes 1-4) or in the presence (lanes 5-8) of KCI. The molar ratio of polymerase/primer in the experiments performed with the intact S. pneumoniae template was 300: 1. Aliquots were removed at 2, 8, 30 and 60 min DNA polymerase I. At a molar ratio of 1: 10 (enzyme/suband analyzed in the gels. SS, single-stranded D N A ; P, position of strate), after 20 s of incubation, only 35% of the polymerized panhandle structures; OC, open circles corresponding to fully replimolecules were elongated by less than 10 nucleotides, and cated M13 DNA. Unlabeled phage T7 D N A digested with MboI was this amount decreased to 8% after 3 min of incubation. In run in parallel as a molecular mass standard; mobility of the T7 bands addition, a significant proprotion of molecules were elongated are indicated to the left of (A) by 29 - 34 (44%) and even 110 - 115 (2%) nucleotides. Furthermore, increased polymerization in the presence of KC1 was detected with the wild-type S. pneumoniae polymerase, as Processivity of the pneumococcal D N A polymerase I expected from the results in Table 3. Arrested polymerization The processivity of the pneumococcal enzymes was esti- after addition of 29 - 34 nucleotides was detected for all three mated by the primer-extension technique according to the enzymes. Nucleotides at this position correspond to the semethod of Tabor et al. [21]. Although processivity is properly quence CCTGTG that has been previously shown to be a defined as the probability of dissociating rather than translo- preferential pause site for the wild-type but not for the Exocating, for practical porposes it can be estimated by length of Klenow fragment [24]. This result was interpreted [24] to be a primer elongation at low enzyme concentration. The results consequence of a hot spot for 3' -+ 5' exonuclease activity, (Fig. 7A) show that the rate of elongation was strongly depen- which caused removal of a nucleotide [(dG),,] from the DNA dent on enzyme concentration for the E. coli Klenow fragment sequence immediately after polymerization. However, this and for the C-terminal fragment of the pneumococcal DNA pause site was detected with the C-terminal fragment of the polymerase I. However, this was not the case for the DNA pneumococcal DNA polymerase I that lacks exonuclease acpolymerase I enzyme; only slight increases in the length and tivity, which indicates that the arrest of polymerization was amount of the labeled fragments were detected at higher molar not due to exonucleolytic degradation, but rather to the DNA ratios of enzyme/DNA primer template. This different pattern sequence surrounding this position, as suggested for other observed with the wild-type pneumococcal polymerase was polymerases [25]. Different patterns of pause sites were detectpresumably not a consequence of its polymerase activity, but ed with different primers complementary to different regions rather of its 5' + 3' exonuclease activity, which could remove of the M13mp2 DNA, but the bands corresponding to an the radiolabel from the 5' end of the longer DNA fragments. elongation of 110- 120 nucleotides remained constant (data This assumption was supported by analysis of the polymeriza- not shown). From the results presented in Fig. 7, it was contion products in agarose gels at longer incubation time or at cluded that S. pneumoniae DNA polymerase I is a polymerase increasing molar ratio of DNA polymerase I enzyme/sub- with intermediate processivity which is able to recycle in the strate. Under these conditions 32P disappeared from the frag- same substrate molecule, and that its C-terminal fragment ments, even though the enzyme was able to extend the primer retains the same processivity and recycling ability. to the full length (7200 nucleotides) of the M13mp2 DNA (results not shown). Strand displacement synthesis catalyzed Fig. 7A shows that, at 1 :I00 and 1:lO molar ratios of by the C-terminalfragment of D N A polymerase 1 DNA polymerase I enzyme/substrate, the elongated DNA molecules were mainly distributed into two positions of about The ability of the enzyme to carry out strand displacement 30 - 34 and 1 10 - 115 nucleotides. Densitometric scanning of was analyzed by performing primer-extension experiments at the gel at 1: 10 molar ratio showed 11 -20% and 10- 18% of a molar ratio of enzyme/primed template of 300:l and the radioactivity at positions 30 - 34 and 110- 115, respective- analyzing the products in alkaline and neutral agarose gels. ly. The C-terminal fragment of DNA polymerase I (at molar Analysis of the samples in a denaturing agarose gel showed ratio 1 : 100) also extended the primer by 110 - 115 nucleotides that an 8-min incubation generated DNA molecules of ap(4% of the polymerized molecules were detected at this po- proximately 7200 nucleotides, corresponding to the entire sition after 3 min of incubation). However, 80% of the length of M13mp2 DNA (Fig. 8A, lane 2). After longer incuelongated DNA molecules increased less than 35 nucleotides bation periods, DNA molecules over 20 000 nucleotides were

155 detected, however the major species observed were about 7200 nucleotides (Fig. 8A, lanes 3 and 4). At high ionic strength, the same pattern was observed (Fig. 8 A, lane 5 - 8), although polymerization was slower. After 2 min of incubation, the major products were approximately 3500 nucleotides long (Fig. 8A, lane 5), whereas at low ionic strength the major species were about 5000 nucleotides (Fig. 8A, lane 1). These results could be ascribed to the lower processivity of the enzyme at high ionic strength (Fig. 7B). Analysis of the samples in neutral agarose gel (Fig. 8B) showed that after 8 min of incubation two major bands were detected, one at the position of the RFII form of the M I 3mp2 and the other a t the position of the panhandle structures (Fig. 8B, lanes 2 and 6). The amount of these structures increased with time of incubation (Fig. 8 B, lanes 3 , 4 , 7 and 8); they are generated from the RFII form by strand displacement followed by strand switching [21, 261. The length of the newly synthesized strand in these structures is only slightly greater than 7200 nucleotides, as shown in Fig. XA. These results show that the C-terminal fragment of the S. pneumoniue DNA polymerase I is able to catalyze strand displacement in an M13mp2 DNA singly primed with a hepta decameric oligonucleotide. Polymerization generates mainly panhandle structures although the enzyme is also able to extend the primer to a length of more than twice the MI 3 genome. In summary, a reliable overproducer of the C-terminal fragment of the S. pneurnoniue DNA polymerase I has been constructed, which contains the polymerase domain of the enzyme. The protein is produced in a soluble and active form. A simple purification scheme has been developed and the enzyme shows polymerase activity with a specific activity of 27 Ujpg protein with activated DNA. This value was close to the 50 U/pg reported for the hyperproduction and purification of the E. coli Klenow fragment [3]. The pneumococcal enzyme lacks exonuclease activities, like the E. coli C-terminal domain of DNA polymerase I [4], but it has the advantage of being produced to a high yield in a soluble form with a normal affinity for the substrates. Like its parental DNA polymerase I, the fragment has an intermediate processivity. It apparently translocates along the substrate for less than 120 nucleotides. The enzyme fully replicates a primed single-stranded M I 3 DNA, and can give rise to strand displacement. These characteristics make the C-terminal fragment of S. pnrunzoniue suitable for 3’ end labeling of DNA fragments and for the conversion of 5’-extended ends to blunt ends without interferences of exonucleolytic activities. In addition, the ability of the enzyme to correctly incorporate nucleotides into DNA, allows its use for DNA nucleotide sequence determination by the dideoxy-chain-termination method. We thank Jeanne Wysocki for carrying out the N-terminal sequence determination. J. J . Dunn and F. W. Studier kindly provided us with the E. coli BL21(DE3)(pET-3) expression system. We thank M . Espinosa for helpful discussions and critical reading of the manuscript. Research at Rrookhaven National Laboratory was under the auspiccs of the US Department of Energy Office of Health and Environmental Rcscarch, and supported by US Public Health Service

grants A114885 and GM29721 from the National Institutes of Health to S. A. L. Research at the Centro dcJInwsti~acioncsBiologicus was under the auspices of the Cons+ Superior de Invrstiguciones Cientificus, and supported by Comision Interministerial de Cienciu J J Twnologia grant BI088-0449 and by Comunidad de Madrid grant (271-90. This work was supported in part by NATO grant 0199-88 to P. L. and S. A. L.

REFERENCES 1 . Kornberg, A. (1980) D N A Replication, W. H . Freeman and Co.,

San Francisco. 2. Klenow, H. & Henningsen, I. (1970) Proc. Nut1 Acud. Sci. U S A 65, 168-175. 3. Joyce, C. M. & Grindley, N. D. (1983) Proc. Nut1 Acad. Sci. LISA 80,1830-1834. 4. Freeniont, P. S., Ollis, D. L., Steitz, T. A. &Joyce, C. M. (1986) Proteins I , 66-73. 5. Martinez, S., Lopez, P., Espinosa, M. & Lacks, S. A. (1986) Gene (Amst.) 4, 79-88. 6. Lhpez, P., Martinez, S., Diaz, A,, Espinosa, M. & Lacks, S. A. (1989) J . Biol. Chem. 264,4255-4263. 7. O h , D. L., Brick, P., Hamlin, R., Xuong, N. G . & Steitx, T. A . (1985) Nature 313, 762- 766. 8. Steitz, T. A,, Beese, L., Freemont, P. S. & Sanderson, M . R. (1987) Cold Spring Harbor Symp. Quunt. Biol.52, 465-471. 9. Studier, F. W., Rosenberg, A. H. & Dunn, J . J. (1989) Methods Enzymol. 185.60 - 89. 10. Rosenberg, A. J., Lade, B. N., Chui, D., Lin. S., Dunn, J. J . & Studier, F. W. (1987) Gene (Amst.) 56, 125-135. 11. Lacks, S. A., Lopez, P., Greenberg, B. & Espinosa, M. (1986) J . M o l . Biol. 192, 753-765. 12. Currier. T. C. & Nester, E. W. (1976) Anal. Biochem. 76, 431 441. 13. Kushner, S. R. (1978) in Genetic enginwring (Boyer, H . W. & Nicosia, S.. eds) pp. 17-23. 14. Birnboim, H. C. & Doly, .I. (1979) Nucleic Acids Res. 7, 15131523. 15. Maxam, A. M. &Gilbert, W. (1980) Methods Enzymol.65,499 560. 16. Studier, F. W . & Moffat, B. A. (1986) J . Mol. B i d . 189, 113130. 17. Laemmli, U. K. (1970) Nature 227,680-685. 18. Rosenthal, A. L. & Lacks. S. A. (1978) J . Biol. Chem. 253, 86748676. 19. Spanos, A., Sedwick, S. G., Yarranton, G. I., Hubscher, U . & Banks, G . R. (1981) Nucleic Acids Res. 9 , 1825-1839. 20. Kunkel. T. A. (1985) J . Biol. Cliem. 260, 5787-5796. 21. Tabor, S., Huber. H . E. &Richardson, C. C. (1987) J . B i d . Cliem. 262, 16212 - 16 223. 22. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 23. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Nut1 Amd. Sci. USA 74, 5463 - 5467. 24. Joyce, C. M. (1989)J. B i d Chem. 264, 10858-110866. 25. Salhi, S., Elie, C., Forterre, P., de Recondo, A . & Rossignol, J . (1989) J . Mol. Biol. 209. 635-644. 26. Lechner, R . L., Engler, M. .I. & Richardson, C. C. (1983) J . Biol. Chem. 268.1 1 174- 11 184.