fVlolecular Microbiology (1992) 6(20), 3009-3019

The 5' to 3' exonuclease activity of DNA poiymerase I is essentiai for Streptococcus pneumoniae Asuncjdn Diaz,^ Sanford A. Lacks^ and Paloma Lbpez^* ^Centro de Investigaciones Biclogicas, CSIC, Velazquez 144. 28006 Madrid. Spain. ^Department of Biology, Brookhaven National Laboratory, Upton, New York 11973, USA.

Summary Three different mutations were introduced in the polA gene of Streptococcus pneumoniae by chromosomal transformation. One mutant gene encodes a truncated protein that possesses 5' to 3' exonuctease but has lost polymerase activity. This mutation does not affect ceii viability. Other mutated forms of polA that encode proteins with only polymerase activity or with no enzymatic activity could not substitute for the wiid-type polA gene in the chromosome unless the 5' to 3' exonuclease domain was encoded elsewhere in the chromosome. Thus, it appears that the 5' to 3' exonuclease activity of the DNA polymerase I is essential for cell viability in S. pneumoniae. Absence of the polymerase domain of DNA polymerase I slightly diminished the ability of S. pneumoniae to repair DNA lesions after ultraviolet irradiation. However, the polymerase domain of the pneumococcal DNA polymerase I gave almost complete complementation of the poiA5 mutation in Escherichia coli with respect to resistance to ultraviolet irradiation.

Introduction The Escherichia coli DNA polymerase I (Eco Poll) encoded by the polA gene is the prototype of type 1 bacteria! polymerases. The biological functions of this multifunctional protein in the processes of DNA replication and repair have been extensively studied (reviewed by Kornberg and Baker, 1992). Eco Poll appears to be responsible for the excision and replacement of RNA at the 5" termini of nascent fragments during semiconservative chromosome replication (Kornberg and Baker, 1992; Wu etai, 1992), In UvrABC-dependent excision repair, Eco Poll, but not the Klenow fragment, yields repair patches

Received 29 April, 1992; revised and accepted 23 June, 1992, "For correspondence. Tel. (1) 585 4203: Fax (1) 562 7518,

that can be sealed by DNA ligase. In addition. Eco Poll and helicase II are absolutely required for the UvrABC complex to incise DNA in a catalytic manner (reviewed by Van Houten, 1990), The 5' to 3' exonuclease activity of Eco Poll also appears to play a major role in the postreplicational repair of ultraviolet lesions (Sharma and Smith, 1987), The Streptococcus pneumoniae DNA polymerase I (Spn Poll) encoded by its pcIA gene is able to substitute in vivo for the E. coti and Bacillus subtilis Poll proteins in DNA repair processes (Lopez et ai, 1987; Martinez et ai, 1987), Spn Poll possesses 5'to 3'exonuclease as well as DNA polymerase activity. These enzymatic domains are respectively located at the A/-terminal (Diaz et ai, 1992) and at the C-terminal (Pons et ai, 1991) regions of Spn Poll. The polA gene of S. pneumoniae was cloned (Martinez et ai, 1986) and its DNA sequence determined (Lopez et ai. 1989). Comparison of the deduced amino acid sequences of the E, coli. S. pneumoniae, and Thermus aquaticus DNA polymerases (Joyce, et ai, 1985; Lopez et ai, 1989; Lawyer et ai. 1989) indicates a common origin for these proteins. However, Taq Poll (Tindall and Kunkel, 1988) and Spn Poll (Diaz etai. 1992) lack the 3' to 5' exonuclease activity detected in Eco Poll {Kornberg and Baker, 1992). To gain more insight into the function of Spn Poll in S. pneumoniae. pcIA mutants were constructed, and their behaviour in DNA repair was examined. Mutations affecting different enzymatic activities were introduced into plasmids containing the wild-type polA gene and linked to a cat marker to allow selection of the mutant polA genes after their introduction into the chromosome by transformation. Of particular interest was the requirement of the 5* to 3' exonuclease activity of Spn Poll for cell viability in S. pneumoniae.

Results Transfer of po\A mutations into the chromosome ofS. pneumoniae strain 641 Three plasmids containing polA mutations that eliminate the polymerase, the 5' to 3' exonuclease, or both Spn Poll activities were used for transfer experiments, in addition to pSM29, which carries the wild-type polA gene. In plasmid pSM40, nucleotides 376 to 1857 of the polA gene are

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A. Diaz, S. A. Lacks and P. Lopez

Table 1. Transfer of cat-polA mutations to the polA' gene in the chromosome of S. pneumoniae strain 641 .^ Transformants ml ^ Donor DNA" pSM29 pSM40 pSM41 pSM51 R6

(Exo' Por) (Exo" Pol") (Exo* Pol") (Exo" Pol')

Cm"

Sm*^

450 000 7 690 000 26 7700

Frequency of transformation (%) 1.09 0,000017 1,68 0,000063 0.02

a. 2.5Mg of plasmid DNA digested with EcoRI and Psfl or 0,5 ng of chromosomal DNA were used to transform 1 ml of competent culture as described in the Experimental procedures. b. Except for R6, which was chromosomal DNA, the other DNAs were plasmids, the polA genes of which conferred phenotypes indicated in parentheses.

replaced by a DNA insert containing a caf gene. As a consequence, the polA gene product (Polln125) should lack both of the Poll activities (see the Experimentat procedures). Plasmid pSM41 carries the potA gene truncated by a cafgene at position 1052, and it encodes a polypeptide (Polln351a) containing the first 351 amino acid residues of Spn Poll and 10 abnormal amino acid residues at its carboxyl-terminus (Diaz et at., 1992), To avoid transcriptional readthrough, the DNA insert used to introduce potA mutations in the aforementioned plasmids carries a transcriptional terminator downstream of the pCi94 cafgene. In addition, the cat-polA junctions present in these plasmids do not generate in-frame fusions. Plasmid pSM51 contains two insertions, at positions 81 and 184, and a deletion trom 184 to 805 of the polA gene. The first insertion includes the cat gene under control of the tet promoter and without transcriptional terminators,

so as to allow transcriptional readthrough from polA and tef promoters. The second insertion introduces a nbosomal binding site and a start codon for translation to generate an in-frame fusion {see the Experimental procedures). which allows synthesis of a polypeptide (Pollc269) containing the polymerase domain of Spn Poll (Diaz et ai, 1992), The cloned chromosomal DNA in the three plasmids containing polA mutations with a cat gene marker was used to transform strain 641. and transformants were selected for chloramphenicol resistance (Table 1). Transformation of the chromosomal insert in pSM29, which contains the cat gene located upstream of and transcribing divergently from the potA^ gene, served as a controi for the efficiency of transformation. The mutant potA gene in pSM41, which encodes only the 5' to 3' exonuclease domain, substituted for the polA* gene in the pneumococcal chromosome with a high efficiency similar to that obtained with pSM29, This result shows that the polymerase domain of Spn Poll is not required for S, pneumoniae cell viability. This polA mutant strain, in which the polymerase of Poll is eliminated, was named MP551 {Table 2), In contrast to the result with pSM41. the levels of transformation with pSM40 and pSM51, which contain polA deletions that eliminate exonuclease activity, were approximately 100000-fold lower than the control. Southern blot analysis of chromosomal DNA from these few transformants revealed rearrangements or duplications within the polA region and retention of an intact potA gene {data not shown). Similar transformation results with these plasmids were obtained when a wild-type strain of S. pneumoniae, R61, was the recipient (data not shown). In addition, the same transformation pattern was observed when glucose replaced sucrose in the selection

Table 2. S. pneumoniae strains used in this study. polA gene product"

Source/ Reference

Wild-type Multiple mutant of R6 potA', malM". end-1, noz-19,exo3

Poll Poll

MP551 MP555

pSM41 -*strain 641 Transformant of 641

MP557

pSM41 ^strain 555

MP558

pSM40 ^strain 555

Polln351a Poll Polln351b Polln351a Polln351b Pollni25 Poi(n351b

Lacks, 1970 Lacks et al. 1975 This work

This work

MP560

pSM51 ^strain 555

Pollc269 Polln351b

This work

Strain

Derivation^

R61 641

Relevant features

poMii(1052bp::ca() polA' ma/A*i(418 bp::(po/A(105 bp)-erm)) product, was detected in strains MP555, MP557, MP558 and MP560 (Fig. 3A, lanes 3-6). The specific proteolytic fragment of Spn Poll containing its /V-terminal region, a 38 kDa polypeptide previously described (Diaz et ai, 1992), was detected by its nucleolytic activity in all strains (Fig.3A,a). Poiymerase activity of polA mutants was measured in crude extracts in vitro (Table 4). Deletion of the poiymerase domain of Spn Poll resulted in a 20-fold decrease in the activity. These results show that Spn Poll is the major DNA polymerase in the bacteria, but they also indicate the presence of another DNA polymerase in pneumococcal cells. Substitution of Spn Pol! by Spn Pollc269 in strain IVIP560 decreased the polymerase activity ascribabie to the Poll enzyme by a factor of five, in agreement with previous results obtained for expression of

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A. Diaz, S. A. Lacks and P. Lopez

i

2

3

4

5

Fig. 3. Gel assays for DNase (A) and DNA polymerase (B) m extracts from S. pneumoniae strains 641 (lane 1), MP551 (lane 2), MP555(lane 3), MP557 (lane 4). MP558 (lane 5) and MP560 (lane 6). Positions of Poll and its derivatives are indicated: a, specific proteolytic fragment of Spn Poll.

6

Poll-

-Polln35ib Polln35la -(a)

Poll -

these enzymes from plasmids, in which the mutant polymerase showed reduced activity (Diaz etai, 1992). Punction of Spn Pel 1 in DNA repair after ultraviolet (u. v.) irradiation The abiiity of various pneumococcai strains to repair DNA was examined (Fig. 4A). Strain MP558 showed greater sensitivity to u.v. than the parental strain MP555. Partial recovery of u.v. resistance, presumably due to DNA repair activity, was observed in strain IVIP560. This complementation by Spn Poilc269 may have been incomplete on account of the lesser polymerase activity of Spn Pollc269 compared with Spn Poll (Table 4). Survival of strain MP555 after u.v. irradiation was lower than that of strain R61 (Fig. 4A) but similar to that of its parental strain 641 (data not shown). It seems, therefore, that the higher sensitivity of strain MP555 is due to genetic factors other than malMi}{4:& bp::a>(po/A(1054 bp)-erm) in its chromosome. We previously detected functional complementation of the E. co//po/-45 mutation (encoding an Eco Poll partially deficient in polymerizing ability) by the pneumococcal

potA gene in DNA repair processes (L6pez et ai. 1987). In this work, we wanted to test whether the individual 5' to 3* exonuclease or poiymerase domains were able to compensate for E. coli polA mutations. Plasmid pSM45 (encoding the Spn Poll poiymerase domain) complemented the DNA polymerization deficiency of the polA5 strain MB1043 almost as efficiently as pSM31, which encodes the entire Spn Poll (Fig. 4B). The presence of plasmid pSM44, which encodes the Spn Poll 5' to 3*

Table 4. DNA polymerase activity in cell extracts from S. pneumoniae strains.

Strain

Polymerase activity^ (U(mg protein)"')

641 MP551 MP555 MP557 MP558 MP560

24,0 1.2 23.4 1.4 1 1 50

a. Each figure is an average of at least three independent determinations; standard deviations were generally