JOURNAL OF BACTERIOLOGY, Mar. 1992, p. 2014-2024

Vol. 174, No. 6

0021-9193/92/062014-11$02.00/0 Copyright © 1992, American Society for Microbiology

Streptococcus pneumoniae DNA Polymerase I Lacks 3'-to-5' Exonuclease Activity: Localization of the 5'-to-3' Exonucleolytic Domain ASUNCION DIAZ,' MARIA ELENA PONS,' SANFORD A. LACKS,2 AND PALOMA

LOPEZ"*

Centro de Investigaciones Biol6gicas, Consejo Superior de Investigaciones Cientificas, Veldzquez 144, 28006 Madrid, Spain, and Biology Department, Brookhaven National Laboratory, Upton, New York JJ9732 1

Received 18 October 1991/Accepted 17 January 1992

The Streptococcus pneumoniae polA gene was altered at various positions by deletions and insertions. The polypeptides encoded by these mutant polA genes were identified in S. pneumoniae. Three of them were enzymatically active. One was a fused protein containing the first 11 amino acid residues of gene 10 from coliphage T7 and the carboxyl-terminal two-thirds of pneumococcal DNA polymerase I; it possessed only polymerase activity. The other two enzymatically active proteins, which contained 620 and 351 amino acid residues from the amino terminus, respectively, lacked polymerase activity and showed only exonuclease activity. These two polymerase-deficient proteins and the wild-type protein were hyperproduced in Escherichia coli and purified. In contrast to the DNA polymerase I of Escherichia coli but similar to the corresponding enzyme of Thermus aquaticus, the pneumococcal enzyme appeared to lack 3'-to-5' exonuclease activity. The 5'-to-3' exonuclease domain was located in the amino-terminal region of the wild-type pneumococcal protein. This exonuclease activity excised deoxyribonucleoside 5'-monophosphate from both double- and singlestranded DNAs. It degraded oligonucleotide substrates to a decameric final product.

The Poll bacterial DNA polymerases encoded by the poL4 genes are multifunctional proteins. The Escherichia coli Poll polypeptide contains three enzymatic domains, two exonucleases, and a polymerase (14). The 5'-to-3' exonuclease is located in the N-terminal region of the protein (10, 12). The remainder of the protein, which corresponds to the Klenow fragment (E. coli PolIK), carries the 3'-to-5' exonuclease and

polymerase domains present in the S. pneumoniae polymerase are arranged in the same order as those in the E. coli and T. aquaticus polymerases. However, despite an earlier indication to the contrary, the S. pneumoniae polymerase, like the T. aquaticus polymerase, apparently lacks 3'-to-5' exonuclease activity. The enzymatic properties of the 5'-to-3' exonuclease activity of S. pneumoniae Poll were characterized.

polymerase domains (13). The Streptococcus pneumoniae polA gene was cloned, and its DNA sequence was determined (25, 27). The S. pneumoniae Poll encoded by this gene was purified and shown to carry polymerase and exonuclease activities (25). S. pneumoniae PolI is able to substitute in vivo for the E. coli and Bacillus subtilis Poll proteins in DNA repair processes (24, 28). In addition, comparison of the amino acid sequences of E. coli, S. pneumoniae, and Thermus aquaticus DNA polymerases (2, 21, 25, 29) indicates a common origin for these proteins. However, the T. aquaticus polymerase, although it contains an associated 5'-to-3' exonuclease activity (22), lacks 3'-to-5' activity (42). The S. pneumoniae and T. aquaticus polymerases are 51 and 96 amino acids shorter, respectively, than the E. coli PolI enzyme. These differences appear to be due to several deletions in the 3'-to-5' exonuclease domain. Inasmuch as the primary structures of the S. pneumoniae, T. aquaticus, and E. coli PolI proteins show some differences as well as considerable similarity, we thought it informative to localize and characterize the enzymatic activities of S. pneumoniae Poll. We constructed derivatives of plasmid pSM22, which contains the cloned S. pneumoniae polA gene, with various mutations in the gene, and we characterized the altered proteins that were produced. Comparison of the enzymatic activities of the mutant S. pneumoniae Poll proteins indicated that the 5'-to-3' exonuclease and *

MATERIALS AND METHODS Bacterial strains and plasmids. The S. pneumoniae strains used were 708 (end-I exo-2 trt-1 he-x-4 malM594) (17) for cloning experiments and 641 (end-i noz-19 exo-3) (18) for characterization of gene products and determination of enzymatic activities. The strains of E. coli used were BL21(DE3) (39) for cloning and expression of enzymes to be purified and strains NR9099 [A(pro-lac) recA ara thil F'(proAB lacI lacZ AM15)], CSH50 [A(pro-lac) ara thil F'(traD36 proAB lacIq lacZ AM15)], and MC1061 [hsdR+ hsdM+ araD A(ara leu) AlacIPOZYgalUgalKstrA], kindly provided by T. A. Kunkel, for experiments with M13mp2 bacteriophage. The bacteriophages used were M13mp2G103 (16), containing a thymine-to-guanine change at position 103, which creates a missense codon at lacZ gene and yields a mediumlight-blue phenotype, and M13mp2A103 (16), containing a thymine-to-adenine change at position 103, which creates a missense codon and yields a very faint-blue phenotype. The plasmids used (Fig. 1) were pSM22 (27), pSM23 (27), pSM29 (28), pET-5 (which is an E. coli expression vector carrying the T7 gene 10 promoter [31]), and pSM10 (which is a recombinant plasmid containing the 3' two-thirds of the pneumococcal polA gene). Plasmid pSM10 was constructed (30) by inserting the 1.8-kb AvaIl-EcoRI DNA fragment of pSM22 into the BamHI site of pET-3b (40). DNA manipulation and transformation procedures. DNA

Corresponding author. 2014

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preparation, manipulation, and analysis were generally carried out by standard methods (33). DNA fragments were separated by electrophoresis on agarose gels and purified by binding to glass milk, according to the specifications of the supplier of GeneClean (BIO-101, Inc.). The plasmid content of the clones was analyzed in alkaline lysates prepared by the method of Birnboim and Doly (3) for E. coli and by a modification of that method (37) for S. pneumoniae. Cultures of S. pneumoniae were grown and transformed as previously described (23). E. coli was grown in LB medium and transformed as described by Sambrook et al. (33). Transformants were selected in agar medium containing tetracycline (1 jig/ml) or chloramphenicol (5 ,ug/ml) for S. pneumoniae and ampicillin (50 ,ug/ml) for E. coli. In some cases transformants for S. pneumoniae 708 were selected by using a previously described DNase colony assay (17). Cells were plated in agar medium containing DNA and methyl green, and the plates were incubated at 37°C for 30 h. Colorless zones (halos) surrounding colonies indicate degradation of DNA by nucleases that leak out of the colonies. After 30 h of incubation, halos only appear when the cells produce elevated levels of nuclease encoded by-the plasmids (27). Construction of derivatives of pSM22. To establish the locations of the different enzymatic domains present in S. pneumoniae PolI, we constructed several derivatives of plasmid pSM22 in which the pneumococcal polA gene was mutated. Mutant proteins that contain carboxyl-terminal or amino-terminal fragments of S. pneumoniae Poll are designated PolIc or PolIn, respectively, with a number appended that corresponds to the first or last S. pneumoniae PolI amino acid residue, respectively, in the mutant protein. To construct plasmids pSM37 and pSM41, we took advantage of plasmid transformation (6, 19). This mechanism allows the introduction by recombination of new genetic information coming from a linear donor DNA molecule into a resident plasmid. The donor DNA must contain a selectable marker and share homology at both ends with the recipient plasmid. To construct plasmid pSM37, plasmid pSM29 was digested with SfaNI, the 5' overhanging ends generated were filled in with E. coli PolIK, and the product was then digested with EcoRI. One of the DNA EcoRISfaNI fragments (fragment A) containing the pC194 cat gene and a piece of the pneumococcal chromosomal insert, including the polA promoter, was purified. Plasmid pSM10 was digested with XbaI, the protruding ends were filled in with E. coli PolIK, and a DNA fragment of 920 bp was purified (fragment B). This DNA fragment contains the ribosome-binding site and the first 11 codons of T7 gene 10 in frame with the pneumococcal poLA gene from the AvaIl site of the gene at position 802 (25). Fragments A and B were blunt end ligated; to eliminate circular molecules, the ligation mixture was digested with EcoRI before transformation of S. pneumoniae 708(pSM22). Transformants were selected for chloramphenicol resistance and examined for the absence of a halo of DNA degradation around the colonies. These transformants contained a plasmid of the expected size (9.2 kb); this plasmid was designated pSM37 (Fig. 1). The mutant poU4 gene present in the plasmid should encode a fused polypeptide (designated PolIc269) composed of 14 abnormal amino acids at its N-terminal region and the carboxyl-terminal 609 amino acids of S. pneumoniae PolI

(Fig. 2).

To interrupt the polA gene at the position of its HindIIl site, plasmid pSM22 was digested with HindIIl and filled in with the E. coli PolIK. This material was treated with BglII

S. PNEUMONL4E DNA POLYMERASE I ACTIVITIES

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and EcoRI, and the 1.65-kb BglII-HindIII and 1.61-kb HindIII-EcoRI fragments (fragments C and E, respectively) were purified. Plasmid pJS3 (1) was cleaved by Sau3AI into two fragments, and the 5' overhangs were filled in with EcoRI PolIK; the 1.12-kb fragment containing the pC194 cat gene under control of the pLS1 tet promoter (fragment D) was purified. DNA fragments C, D, and E were ligated together and digested with EcoRI. The ligation was used to transform S. pneumoniae 708(pSM22); because of the homology provided by fragments C and E, the cat gene was introduced into the HindIII site of pSM22. The resulting plasmid was designated pSM41 (Fig. 1); it contains a mutant poL4 gene, which should encode a polypeptide (called S. pneumoniae PolIn351) containing the first 351 amino acids of S. pneumoniae Poll and 10 abnormal amino acids located at its carboxyl terminus (Fig. 2). To eliminate the central portion of thepoLA gene, plasmid pSM29 was digested with TthlllI and BamHI; after the cohesive ends were filled in, the 8.7-kb fragment was recircularized by ligation. The resulting plasmid was called pSM38 (Fig. 1); its polA gene product predicted from the DNA nucleotide sequence is S. pneumoniae Polln264, a fused polypeptide composed of the amino-terminal 264 amino acids of S. pneumoniae PolI and 15 anomalous amino acids at its carboxyl terminus (Fig. 2). The pneumococcal polA gene was interrupted at the position of the unique BamHI site of pSM22. To this end, pSM22 was linearized with BamHI and ligated to the 1.12-kb Sau3AI fragment of pJS3 containing the cat gene. The resulting plasmid was called pSM28 (Fig. 1); its polA gene should encode the mutant S. pneumoniae PolIn620, composed of the first 620 amino acids and 7 abnormal amino acids at its carboxyl terminus (Fig. 2). The EcoRI inserts containing the mutated poLA genes present in plasmids pSM38, pSM41, and pSM28 were subcloned into the unique EcoRI site of the E. coli pET-5 vector. The resulting plasmids, with the insertion in the correct orientation for expression, were designated pSM380, pSM410, and pSM280 (Fig. 1). DNA sequence analysis. The DNA nucleotide sequence at the fusion junction within the mutant polA genes present in plasmids pSM28, pSM37, pSM41, and pSM380 was determined by the dideoxy chain terminator method of Sanger et al. (34) with an alkaline-denatured double-stranded plasmid (5) annealed to the appropriate complementary primers by using [a-32P]dATP and a T7 polymerase kit (Pharmacia). To sequence pSM28, pSM37, pSM41, and pSM380, respectively, the following synthetic oligonucleotide primers, purchased from Biogen, were used (nucleotide numbers refer to positions relative to the fusion junctions): TTGGCTTG CAGGACTGG (nucleotides 77 through 93), GCCACAT CAGCTGACGA (nucleotides 75 through 91), GCTGAGT GAAGAGTCTA (nucleotides 98 through 114), and CAATGA CAAGGAACAGG (nucleotides 68 through 84). Preparation of cell extracts. To prepare crude extracts for in vitro enzymatic assays, cultures of S. pneumoniae were grown in casein hydrolysate medium to an optical density at 650 nm of 0.5. Cells from 1.5 ml were sedimented by centrifugation, washed by suspension in 1 ml of buffer A (10 mM Tris-HCl [pH 7.6], 50 mM NaCl), centrifuged again, and suspended in 150 ,ul of buffer A. Cells were lysed by the addition of 0.05% Triton X-100 and 15 min of incubation at 30°C. The viscosity of the extracts was reduced by passage through a 0.36-mm-inner-diameter needle. To assay enzymatic activities in gels, cell extracts were prepared as previously described (27).

2016

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PLASMID VECTORS I 1

INSERT

polA

I~~~~ Z

m

l

EcoRi

EcoRI

00

o E

-4 *CO1 Cs

w

pSM22 'a

I

pSM23

pSM29

cat

pSM38

p

pSM41

pSM4I0

pSM28

pSM280

SM380

cot

cat

cat

pSM37 cat

S.D.

FIG. 1. Physical maps of the inserts in plasmid pSM22 and its derivatives. Genes are transcribed in the directions indicated by the arrows. poL4 encodes S. pneumoniae Poll, tet encodes tetracycline resistance, and cat encodes CAT. S.D., ribosome-binding site for translation of T7 gene 10. P+10, promoter for transcription of T7 gene 10. Dashed lines indicate deletions within the poU gene. Only relevant restriction sites are depicted. The names of the plasmids obtained by placing the inserts into two different vectors are given on the right.

For protein purification, 500-ml cultures of BL21(DE3) carrying plasmid pSM23, pSM280, or pSM410 were grown in M9 medium supplemented with ampicillin (200 ,ug/ml) at 37°C to an optical density at 600 nm of 0.5; at this time isopropyl-13-D-thiogalactopyranoside (1 mM) was added. After 1 h of incubation at 37°C, rifampin (200 ,g/ml) was added, and the cultures were incubated for 1 h more. Cells were harvested by centrifugation and concentrated 100-fold in 0.5 M NaCI-10 mM Tris-HCl (pH 7.6)-3 mM P-mercaptoethanol-1 mM EDTA. Crude extracts were prepared by passage through a French pressure cell at 20,000 lb/in2. All further steps were carried out at 4°C. Cell debris was removed by

centrifugation at 15,000 x g for 30 min. The supernatant fluids are designated as the crude extracts (Table 1). Streptomycin sulfate was added to the crude extracts to a final concentration of 5.8%. The suspension was allowed to stand 30 min and then centrifuged at 15,000 x g for 30 min. Ammonium sulfate (75% saturation) was added to the supernatant fluids (designated as streptomycin sulfate supernatants in Table 1). The resulting precipitates were dissolved in buffer A and were designated as ammonium sulfate extracts (Table 1). Purification of wild-type and polymerase-deficient S. pneumoniae PolI proteins expressed in E. coli. Strains of

Poll

1 MDXX ... APIAIGL3DLVYSGPDVNLGKFYD 279 ... ATDKLBLLQDPIFXD 361 ... RLSSVDPNLQNIP 627 ...

Poln264

1 MDKX

PolIn35l

1

PolIn620

1 MDXX .................... RLSSVDLCYrKt 627

DXX

...

APIAIGLEDLI lX

iPDW*

WTUX

877

279

.................................

ATDXLIZPVX28Kf

361

. 623 .... E55M!lGwDE pDVENLGxFYD FIG. 2. Amino acid sequence alignment of wild-type and mutant S. pneumoniae Poll. Amino acid sequences predicted from the DNA nucleotide sequences of the amino-terminal, carboxyl-terminal, and fusion junction regions are depicted. Numbers indicate the amino acid positions relative to the N-terminal end of each DNA polymerase. The abnormal amino acids present in the altered S. pneumoniae Poll are indicated in italic boldface type. Dotted lines indicate identical amino acids in the wild-type and mutant polypeptides.

PolIc269

1

.....................................................

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TABLE 1. Purification of S. pneumoniae IPoll, PolIn620, and PolIn351 proteins

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gradient (50 to 550 mM) of NaCl in the same buffer. Again, a single peak of polymerase activity (eluting at 0.15 M NaCI) Protein Exoriuclease was obtained. The peak was ascribed to S. pneumoniae Poll, % Yielda Sample and fraction (mg) activit a 99-kDa polypeptide visualized in a Coomassie blue-stained y (U/mg) sodium dodecyl sulfate (SDS)-polyacrylamide gel (Fig. 3A, Poll lane 5) and in a nuclease activity gel (Fig. 3B, lane 5). The 222 100 Crude extract 77.9 polymerase peak was diluted fourfold and applied to a Streptomycin sulfate 156 75.8 DEAE-Sephacel column (15 by 0.9 cm; Pharmacia). The supernatant column was developed with a linear gradient of NaCl (50 to Ammonium sulfate 276 43.3 69.1 550 mM); S. pneumoniae PolI eluted at 0.26 M NaCl. A extract Bio-Gel A-0.Sm 12.0 503 summary of the purification is presented in Table 1 and Fig. 35.0 fractions 48 3A and B. The purified preparation of S. pneumoniae PolI through 56 showed less than 10% impurities by Coomassie blue staining Heparin-agarose 1.6 2 ,340 21.8 (Fig. 3A, lane 6), and it had nuclease activity (Fig. 3B, lane fractions 36 6). Slight contamination with its specific proteolytic product through 41 was detected as nuclease activity of the protein in the gel (25) DEAE-Sephacel 15.9 0.8 3 ,250*25 15. at 34 kDa (Fig. 3B, lane 6). Partial conversion of the intact fractions 42 Poll to its proteolytic product was observed upon storage of through 45 the enzyme preparation in 50% glycerol at -20°C. S. pneumoniae PolIn620 was purified from BL21(DE3) PolIn620 Crude extract 226 75.0 100 (pSM280) by agarose gel filtration. Two peaks of nuclease 73.5 Streptomycin sulfate 204 88.4 activity were detected. The first peak (fractions 48 through supernatant 56) was ascribed to the expected S. pneumoniae PolIn620, a Ammonium sulfate 35.6 415 87.1 71-kDa polypeptide, from the nuclease position in the gel extract Bio-Gel A-0.Sm 10.1 528 assay (Fig. 31.4 3D, lane as 4).detected The second corresponded 25-kDa polypeptide withpeak a nuclease activitytogela fractions 48 (data not shown). The first peak was and dialyzed applied to through 56 Heparin-agarose 0.9 11.8 a heparin-agarose column. Bound proteins were eluted with 2 ,120 a 100-ml linear gradient of NaCl (10 to 310 mM). A single fractions 38 through 42 peak of nuclease activity (eluting at 0.10 M NaCl) was DEAE-Sephacel 0.5 2 ,350 7.2 diluted and chromatographed on DEAE-Sephacel. The colfractions 33 umn was developed with a linear gradient of NaCl (100 to 600 through 38 mM). Fractions 33 through 38 contained the S. pneumoniae PolIn620 polypeptide and a 65-kDa polypeptide (Fig. 3C, PolIn351 Crude extract 57.0 350 100 lane 6); both proteins showed nuclease activity (Fig. 3D, Streptomycin sulfate 55.1 291 80.3 lane 6). Because the 65-kDa protein was not detected in the crude extracts, we assume that it was generated by proteosupernatant Ammonium sulfate 513 27.3 70.2 lysis of the S. pneumoniae PolIn620 fragment during purifiextract cation (Fig. 3D, lanes 1 through 3). In addition, conversion Bio-Gel A-0.5m 13.1 397 26.1 of S. pneumoniae PolIn620 to the 65-kDa polypeptide was fractions 53 observed upon storage of the preparation in 50% glycerol at through 60 -20°C. A summary of the purification is presented in Table Cellex P fractions 12 3.0 767 11.7 1 and Fig. 3C and D. through 17 S. pneumoniae PolIn351 was purified from BL21(DE3) Heparin-agarose 0.5 2 ,350 6.0 (pSM410) by agarose gel filtration. Two peaks of nuclease fractions 31 activity were detected. The first peak (fractions 53 through through 35 60) was ascribed to the S. pneumoniae PolIn351 fragment, a The yields of exonuclease activity in crude ext racts were 17,300 U for 41-kDa polypeptide, on the basis of the nuclease position in PolI, 16,960 U for PolIn620, and 19,960 U for PolIn 351. the gel assay (Fig. 3E, lane 4). Fractions 53 through 60 were pooled, dialyzed, and applied to a 10-ml Bio-Rad Bio-Gel 15by 0.9-cm Cellex P column. The mutant protein did not bind to the column. The six most active fractions were pooled and BL21(DE3) carrying pSM23, pSM280, or pSM410 were chosen as sources of enzymes for purific ation. The cultures chromatographed on heparin-agarose. Bound proteins were were induced with isopropyl-13-D-thioga lactopyranoside in eluted with a 100-ml linear gradient of NaCl (10 to 260 mM). the presence of rifampin, and ammoniuim sulfate extracts A single peak of nuclease activity was detected (eluting at were prepared as described above. 0.25 M NaCl). Analysis of this peak by SDS-polyacrylamide Wild-type S. pneumoniae PolI was p)urified from BL21 gel electrophoresis revealed several polypeptides (Fig. 3E, (DE3)(pSM23). The ammonium sulfate e xtract was applied lane 6). Correlation between the 41-kDa polypeptide and the to an agarose column (Bio-Gel A-O.5r n, 85 by 1.6 cm; nuclease activity was obtained by the gel activity assay (Fig. Bio-Rad Laboratories) and eluted with column buffer (10 3F, lane 6). A summary of the purification is presented in mM Tris-HCI [pH 7.6], 1 mM dithiothre itol, 1 mM EDTA, Table 1 and Fig. 3E and F. 5% ethylene glycol) supplemented with 0.5 M NaCl. The Enzymatic assays. To test for exonuclease activity during single peak of polymerase activity dete:cted was dialyzed enzyme purification, the substrate was salmon sperm DNA against the column buffer supplemented with 50 mM NaCl (previously nicked with pancreatic DNase I) labeled with E. and applied to a Bio-Rad 15- by 0.9-c m heparin-agarose coli PolIK and [3H]dTTP. Exonuclease activity was detercolumn. Bound proteins were eluted wiith a 100-ml linear mined as previously described (25). One unit of exonuclease

6804

a

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DIAZ ET AL.

J. BACTERIOL.

A

C

sI1 23 45 6 -

0:

s

2

E

3

4

5

S 1

6

2 3

4 5 6

a

a

000 '00+

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B

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23456 : t VA

D 4

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FIG. 3. Purification of S. pneumoniae PolI (A and B), PolIn620 (C and D), and PolIn351 (E and F): SDS-12% polyacrylamide gel stained with Coomassie blue (A, C, and E) or developed for nuclease activity in the presence of 2 mM MnCl2 as previously described (32) (B, D, and F). Lanes: S, standard proteins (bovine serum albumin [67 kDa], chicken ovalbumin [43 kDa], bovine carbonic anhydrase [30 kDa], and soybean trypsin inhibitor [20 kDa]); 1, crude extract; 2, streptomycin sulfate supernatant; 3, ammonium sulfate extract; 4, peak of gel filtration column; 5 (A, B, C, and D) and 6 (E and F), peak of heparin-agarose column; 5 (E and F), peak of Cellex P column; 6 (A, B, C and D), peak of DEAE-Sephacel column. Appropriate volumes of each sample were applied onto the gel to give 8 ,ug (A, lanes 1 through 4; C and E, lanes 1 through 5), 4 p,g (C and D, lanes 6), or 3 pg (D, lanes 5 and 6) of protein or 3 U (B, lanes 1 through 6, and D, lanes 1 through 4), 2 U (D, lanes 5 and 6), 1.5 U (F, lanes 1 through 5) or 0.4 U (F, lane 6) of exonuclease activity. Arrows indicate the positions of S. pneumoniae Poll (99-kDa polypeptide) (A and B) and S. pneumoniae PolIn351 (41-kDa polypeptide) (E and F). The positions of the S. pneumoniae PolIn620 (71-kDa polypeptide) (a) and its 65-kDa putative proteolytic fragment (b) are indicated (C and D).

activity corresponds to 10 nmol of nucleotide released from DNA in 30 min at 37°C. To assay the exonuclease activity of purified enzymes, the 17-mer oligonucleotide universal primer (GTAAAAC GACGGCCAGT) was labeled at its 5' end and annealed to M13mp2 single-stranded DNA as described by Tabor et al. (41). It was labeled at its 3' end (after annealing) by treatment with Sequenase version 2.0 (Pharmacia), dGTP, and [a-32P]ddATP to generate a 3'-end-labeled 19-mer oligonucleotide. For the exonuclease assay the reaction mixture (50 pl) contained 22 fmol of DNA substrate, bovine serum albumin (0.5 mg/ml), 10 mM Tris-HCl (pH 7.6), 3 mM 3-mercaptoethanol, and 5 mM MnCl2. To assay the exonuclease activity of E. coli PolI, 10 mM MgCl2 was used instead of MnCl2. The reaction mixture was preincubated at 37°C for 1 min, and the reaction was initiated by adding the enzyme (5 ,ul) diluted in 10 mM Tris-HCI (pH 7.6)-3 mM ,B-mercaptoethanol-0.5 mg of bovine serum albumin per ml. Reactions were stopped by adding 50 mM EDTA. For analysis on denaturing gels, 5 ,ul of the reaction mixture was added to 5 pl of 80% formamide-20 mM EDTA-0.5% SDS-0.1% tracking dyes, heated at 95°C for 2 min, and loaded onto a 20% polyacrylamide gel containing 7 M urea. Quantification of the bands was performed by soft laser

densitometric scanning of the gel autoradiograms in an LKB Ultroscan 2202 scanner coupled to an Apple II computer. To test proofreading activity, we used the terminal mismatch excision assay described by Kunkel et al. (16). DNAs from two mutant derivatives of bacteriophage M13mp2, containing a single base difference at position 103 in the lacZa gene, were used to construct two different doublestranded heteroduplex molecules containing single base mismatches at different distances from a gap of several hundred nucleotides in one DNA strand. These molecules contain a cytosine residue in the primer (minus) strand (which encodes a medium-blue plaque phenotype) opposite an adenine residue in the template (plus) strand, which yields a faint-blue

plaque phenotype. The first heteroduplex contains a 363-nucleotide stretch of single-stranded DNA from the KpnI-to-AvaHI sites of M13mp2, and the mismatch is located at the 3' terminus of the gapped strand. The second heteroduplex contains a 316-nucleotide stretch of single-stranded DNA from the EcoRI-to-AvaII sites of M13mp2, and the mismatch is internally located at 47 nucleotides from the 3' terminus. The gapped molecules were purified by agarose gel electrophoresis. Samples of 0.3 p,g were treated with 2.5 U of the appropriate DNA polymerase in a volume of 50 pl to fill in

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TABLE 2. DNA polymerase and CAT activities in cell extractsa

A

kDa Plasmid -99 -71

-41

B 2 3 4 5 6

^L*|*^f-99 000 t;-71

FIG. 4. Gel DNase (A) and polymerase (B) assays of extracts from S. pneumoniae 641 without plasmids (lane 1) or with plasmids pSM38, pSM41, pSM28, pSM29, and pSM37 (lanes 2 through 6, respectively). Total cell extracts containing 50 p,g of protein were loaded in an SDS-10% polyacrylamide gel. After polyacrylamide gel electrophoresis and removal of the SDS, the nuclease gel was incubated in the presence of 2 mM MnCI2 for 10 days and stained with ethidium bromide. The polymerase gel was incubated with 0.38 nM [a-32P]dTTP (3,000 Ci/mmol) and 12 ,uM each dATP, dGTP, and dCTP in the presence of 7 mM MgCl2 (36) for 20 h. After drying, the gel was exposed for autoradiography for 24 h. The sizes of thepolA gene products are indicated on the right.

the gap. From each reaction a sample of 25 ,ul was analyzed by electrophoresis in a 0.8% agarose gel. All reactions generated products that migrated at the position of completely double-stranded RFII DNA (data not shown). A second sample of 8 ,ul was used to transform E. coli MC1061. The transformation mixture was then plated together with cells of E. coli CSH50, and the resulting M13 plaques were scored for color (15). Protein concentration was determined in crude extracts by the method of Lowry et al. (26) and after partial purification by measurement of the A280 with correction for A260. SDSpolyacrylamide gel electrophoresis was carried out as described previously (20). Gels were stained with Coomassie brilliant blue R260.

RESULTS Identification of poL4 gene products expressed by mutant derivatives of pSM22 in S. pneumoniae. Crude extracts of S. pneumoniae 641 harboring various plasmids were analyzed for nuclease (Fig. 4A) or polymerase (Fig. 4B) activities in situ after SDS-polyacrylamide gel electrophoresis. All of them showed one band with nuclease activity at the position of 99 kDa (Fig. 4A), which corresponds to the position of the S. pneumoniae PolI encoded by the polA4+ gene, present as one copy in the chromosome of these strains, as previously observed (27). This band was more intense in the extract from 641(pSM29) (Fig. 4A, lane 5) because of thepoA + gene in the multicopy plasmid. Another nuclease band was detected in the extract from strain 641(pSM28) (Fig. 4A, lane 4) at 71 kDa, the expected molecular mass for the S. pneumoniae PolIn620 polypeptide. Plasmid pSM41 encoded a

None pSM38

pSM41 pSM28 pSM37 pSM29

Enzyme activityb (U/mg of protein) CAT DNA polymerase

24 22 25 24 111 324

551 498 553 493 540

a Crude extracts were prepared as described in Materials and Methods. Polymerase and CAT activities were determined in vitro as previously described (28). b One unit of polymerase activity is defined as the amount of enzyme catalyzing the incorporation of 1 nmol of nucleotide into DNA per h at 30'C. One unit of CAT activity is expressed as the amount of enzyme catalyzing the acetylation of 1 nmol of chloramphenicol per min. Each value is an average of at least three independent determinations; the standard deviation was generally -J20

5 2.50

00 7.5

~D0

I0 0 2.5

5

7.5

10

TIME (min) FIG. 5. Exonuclease activities of the proteins encoded by the wild-type and mutantpolA genes in S. pneumoniae. Crude extracts (0.75 pug) of pneumococcal strain 641 without plasmids (l) or with plasmid pSM29 (0), pSM28 (0), pSM41 (A), pSM38 (A), or pSM37 (-) were incubated at 37°C with 25 ng of a 2-kb 5'-end-labeled [32P]DNA fragment (12,500 cpm). At the times indicated, the release of soluble radioactivity was measured. Reactions were performed as previously described (25).

with both substrates (Fig. 6, lanes 1 through 10); its major reaction product was deoxyribonucleoside 5' -[32P]monophosphate, but labeled dimer and trimer oligonucleotides were also detected. Such short oligonucleotides are also released by the 5'-to-3' exonuclease activity of E. coli Poll (14). When S. pneumoniae Poll was tested under a variety of conditions, such as different amounts of enzyme, different

tric

FIG. 6. Exonuclease activity of S. pneumoniae Poll on doubleand single-stranded DNA substrates. Assay mixtures consisted of 22 fmol of 5'-end-labeled [32P]17-mer coupled with M13mp2 (doublestranded) substrate (lanes 1 through 5 and 11 through 15) or 5'-end-labeled [32P]17-mer (single-stranded) substrate (lanes 6 through 10 and 16 through 20) and 0.02 U of S. pneumoniae Poll (lanes 1 through 10) or 0.06 U of E. coli Poll (lanes 11 through 20) under the conditions indicated in Materials and Methods. Incubations were performed at 37°C for 2.5 min (lanes 1, 6, 11, and 16), 10 min (lanes 2, 7, 12, and 17), 30 min (lanes 3, 8, 13, and 19), 1 h (lanes 4, 9, 14, and 19), and 2 h (lanes 5, 10, 15, and 21); lane C, 17-mer substrate incubated without enzyme. The positions of the 17-mer substrate and monomer product are indicated on the left.

VOL. 174, 1992

S. PNEUMONAE DNA POLYMERASE I ACTIVITIES

2021

TABLE 3. Absence of a proofreading exonuclease activity associated with the pneumococcal PolI proteina Time of reaction

Plaques scored

% Retention of mismatched bases

Total

Med bldue

Complete heteroduplex"

1,235

750

60.7

Gapped heteroduplex

3,724

91

2.4

1,897 859 1,543 1,620 1,033 852 1,152 1,568

147 340 796 264 256 35 52

7.7 39.6 50.1 49.1 25.5 30.0 3.0 3.3

2,410 2,844

884 265

36.6 9.3

Enzyme

Terminal mismatch E. coli PolIK E. coli PolIK S. pneumoniae Poll S. pneumoniae PolI S. pneumoniae PolI S. pneumoniae PolI S. pneumoniae PolI S. pneumoniae PolI Internal mismatch S. pneumoniae Poll S. pneumoniae PolI

Cation

Mg2+ Mg2+

dNTP

(pM)

dTMP

(RM)

(min)

Mg2+ Mn2+ Mn2+ Mn2+ Mn2+

10 10 10 10 1,000 1,000 1,000 1,000

0 20 0 0 0 20 20

15 15 5 15 5 5 15 15

Mn2+ Mn2+

1,000

1,000

0 0

5 15

Mg2+

0

773

a Reaction mixtures (50 ,ul) contained 45 mM Tris-HCI (pH 8), 2 mM dithiothreitol, 6.5 mM MgCI2 or 5 mM MnCI2, each of the four deoxynucleoside triphosphates (dNTPs) at the indicated concentrations, 2.5 U of S. pneumoniae Poll or E. coli PolIK, and 300 ng of gapped M13mp2 DNA containing the A-C mismatch terminally or internally. b M13mp2 double-stranded DNA containing the A-C internal mismatch and a single nick at the position of the AvaII site.

pHs (pH 5, 8, or 10), Mg2+ instead of Mn2+, and a DNA substrate containing a 3'-end terminal mismatch, similar results were obtained (data not shown). This assay therefore indicates either the absence of 3'-to-5' exonuclease activity in the pneumococcal enzyme or levels less than 6% of that present in E. coli Poll, since no activity was detected even with 1 U of exonuclease in the assay (data not shown). Furthermore, when we tested the 3'-to-5' exonuclease of S. pneumoniae Poll (2 U of polymerase activity) on a 1.6-kb 3'-end-labeled DNA (4 pmol), after 10 min of incubation 3% of the radioactivity was hydrolyzed (data not shown). This result indicates that the ratio of 3'-to-5' exonuclease activity to polymerase activity for S. pneumoniae PolI was