Mutation Research, 235 (1990) 195-201 DNA Repair
195
Elsevier MUTDNA 06382
Excision-repair capacity in Streptococcuspneumoniae: cloning and expression of a uvr-like gene N. Sicard 1 a n d A.M. E s t e v e n o n 2 I Centre de Recherche de Biochimie et de Gdndtique Cellulaires du CNRS, 31062 Toulouse Cedex (France) and 2 U.F.R. Sciences Pharraaceutiques, Unioersitd Paul Sabatier, 31062 Toulouse Cedex (France)
(Received 14 August 1989) (Revision received 29 November 1989) (Accepted 30 November 1989)
Keywords: Streptococcus pneumoniae; Excision repair; Uvr-like gene
Summary Although deficient in photoreactivation and some SOS-like functions, Streptococcus pneumoniae has the capacity to carry out excision repair when exposed to U V light. The repair ability and sensitivity to UV irradiation or treatment with chemical agents in the wild type and a UV-sensitive mutant strain indicate that UV-induced pyrimidine dimers might be repaired in pneumococcus by a system similar to the uvr-dependent system in Escherichia coll. A gene complementing the mutation conferring UV sensitivity of the mutant strain has been cloned. The coding region directs the synthesis of a polypeptide with a molecular weight of 78 kDa. The relationship with uvr-like protein in E. coli is discussed.
The bacterial response to D N A damage caused by UV light, ionizing radiations and chemical agents involves the repair of D N A by several processes (Sancar and Sancar, 1988). It has been reported that Streptococcus pneumoniae is deficient in photoreactivation (Goodgal et al., 1957) and some SOS-like functions (Gasc et al., 1980). We have used a rapid assay that we have developed in our laboratory and which allows detection of pyrimidine dimer excision in a plasmid in vivo after UV irradiation and post-incubation of the host cells. We have shown that S. pneumoniae has
Correspondence: Dr. N. Sicard, Centre de Recherche de Biochimie et de G~n&ique Cellulaires du CNRS, 118 route de Narbonne, 31062 Toulouse Cedex (France).
the capacity to carry out excision repair after exposure to UV light (Estevenon and Sicard, 1989). Excision repair can be achieved by a specific D N A glycosylase and AP endonuclease as observed in Micrococcus luteus (Carrier and Setlow, 1970; Haseltine et al., 1980) or by the uvrABC excinuclease pathway as observed in Escherichia coli (Seeberg et al., 1976; Yeung et al., 1983; Sancar and Rupp, 1983) and also in M. luteus (Tao et al., 1987; Shiota and Nakayama, 1988, 1989). We have studied the mechanism of pyrimidine dimer excision repair in S. pneumoniae by the analysis of the repair ability and sensitivity to UV irradiation and chemical agents in the wild type and a UV-sensitive mutant strain. We have cloned the gene which complements the mutation conferring radiosensitivity to this mutant strain and identified the encoded protein.
0921-8777/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
196 Material and methods
Bacterial strains and plasmid S. pneumoniae R800 was used as the parental strain (Lefrvre et al., 1979). Strain R402 is a UV-sensitive mutant, isolated after transfer of the UV-sensitive mutation 402 (Tiraby and Sicard, 1973) into R800. Cultures of S. pneumoniae were grown at 37 ° C in complete medium (Prats et al., 1985). Plasmid pSP2 was used as the cloning vector in pneumococcus (Prats et al., 1985). Determination of excision-repair capability The method has been previously described (Estevenon and Sicard, 1989). Chemicals cis-Diamminedichloroplatinum (cis-platinum) and 4-nitroquinoline-l-oxide (4NQO) were obtained from Dr. B. Salles (CNRS, Toulouse) and Dr. J. Alonso (Max Planck Institute, Berlin) respectively. Mitomycin C was purchased from Sigma Chemical Company. Restriction enzymes Restriction endonucleases and T4 D N A ligase were obtained either from Bethesda Research Laboratory Inc. (Gaithersburg, MD) or New England Biolabs, Inc. (Beverly, MA) and used as specified by the supplier. UV endonuclease from M. luteus was purchased from Applied Genetics Inc. (Freeport, NY). Survival experiments after irradiation and chemical treatments Exponentially growing cells (5 × 1 0 7 cells/ml) were harvested, washed and resuspended in the mineral buffer of the synthetic medium (Sicard, 1964). Samples of 5 ml were irradiated with UV from a G 1518 Sylvania germicidal lamp producing an incident dose rate of 2 j / m 2 / s . After irradiation, the cell suspensions were diluted and plated on agar medium. Cell survival was determined after overnight incubation at 37 o C. Samples of 5 ml were treated with mutagens as follows: after addition of chemical agents at various concentrations, the samples were kept in the dark for 1 h at 30 o C for mitomycin C and 4NQO,
and 1.5 h for cis-platinum assays. Cell survival was estimated as described above.
DNA isolation and manipulation Total D N A was isolated by the procedure of Lefrvre et al. (1979). Large-scale preparation of pSP2 plasmid was carried out according to IshHorowicz and Burke (1981) except that pneumococcal cells were lysed with a mixture of desoxycholate and sodium dodecyl sulfate (Lefrvre et al., 1979). Plasmid D N A was further purified by cesium chloride ethidium bromide density gradient centrifugation. Products from restriction digestions were analyzed on agarose gels and recovered from the gel by electroelution. D N A transformations were performed as described by Lef~vre et al. (1979). Analysis of plasmid-encoded proteins Purified recombinant plasmid D N A s were transcribed and translated according to the method of Zubay (1973). The products of the synthesis were analyzed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) (Laemmli, 1970). Results and discussion
Sensitivity to UV rays and repair capability of R800 and R402 The survival curves after irradiation of the wild-type strain R800 and its UV-sensitive derivative R402 are shown in Fig. 1. R402 exhibits a high sensitivity to UV rays. To test the repair capability of the mutant strain R402, we used a rapid assay developed in our laboratory (Estevenon and Sicard, 1989) providing an estimate of pyrimidine dimer excision repair in vivo in bacterial strains. It is based on the fact that UV endonuclease converts covalently closed circular (CCC) D N A containing pyrimidine dimers to the open circular (OC) form. Therefore pyrimidine dimer excision repair was revealed by the appearance of CCC plasmid D N A which was no longer sensitive to UV endonuclease. For the assay wild-type and mutant strains were transformed with pSP2 plasmid. The cells were exposed to UV light and incubated for 1 h in complete medium for recovery. Plasmid D N A was isolated,
197
treated with UV endonuclease and analyzed by agarose gel electrophoresis. In the wild-type strain, when the D N A was examined immediately after irradiation, all the D N A was converted to the OC form by UV endonuclease, for an incident dose of 20 or 40 J / m 2 (Fig. 2A, lanes 4 and 8, respectively). However, when post-incubation of 1 h was allowed after irradiation, a small fraction of the CCC D N A was resistant to UV endonuclease showing that some repair had taken place (Fig. 2A, lanes 6 and 10). By contrast, in the mutant strain there was no CCC D N A which was insensitive to UV endonuclease after post-incubation (Fig. 2B, lanes 6 and 10). This shows the absence of excision-repair capacity in the mutant strain R402.
10
al
¢. Q Q.
0.01
I
0
,
5
i"
q
10
20
4'0 tlV
( j/r.2 )
Fig. 1. Effect of UV rays on survival of Streptococcus pneumoniae. • R800 (wild type); o R402 (UV-sensitive mutant); * R402 (pSP800).
_
+,
Sensitivity to chemical agents Pyrimidine dimers might be removed by different incision mechanisms (Sancar and Sancar, 1988). In M. luteus the D N A glycosylase involves the specific recognition of the pyrimidine dimer (Haseltine et al., 1980) whereas the uvrABC excinuclease system in E. coil removes, through nucleotide excision repair, the U V - i n d u c e d pyrimidine dimers as well as the adducts produced
_,
_
OC
OC
CCC
CCC
A
2
3
4
5
6
7
8
9
B
Fig. 2. Incision of damaged pSP2 D N A extracted from UV-irradiated S. pneumoniae R800 (A) and R402 (B). The plasmid pSP2 has been either digested ( + ) or not digested ( - ) with UV endonuclease from M. luteus. Lanes 1-2: plasmid extracted from unirradiated bacteria. Lanes 3-4: irradiated 20 J / m 2 (A), 10 J / m 2 (B); no post-incubation. Lanes 5-6: irradiated 20 J / m 2 (A), 10 J / m 2 (B); post-incubated 1 h. Lanes 7 - 8 : irradiated 40 J / m 2 (A), 20 J / m 2 (B); no post-incubation. Lanes 9-10: irradiated 40 J / m 2 (A), 20 J / m 2 (B); post-incubated 1 h.
198 100,
.,..I
10-..,
1¢ tU O: tU 0,.
o 0,1
o
0,1
o',2
MI TOMYCINE
o',3
C (Hg/ml)
o go ~oo
2bo
4- NQO
~oo
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195
Elsevier MUTDNA 06382
Excision-repair capacity in Streptococcuspneumoniae: cloning and expression of a uvr-like gene N. Sicard 1 a n d A.M. E s t e v e n o n 2 I Centre de Recherche de Biochimie et de Gdndtique Cellulaires du CNRS, 31062 Toulouse Cedex (France) and 2 U.F.R. Sciences Pharraaceutiques, Unioersitd Paul Sabatier, 31062 Toulouse Cedex (France)
(Received 14 August 1989) (Revision received 29 November 1989) (Accepted 30 November 1989)
Keywords: Streptococcus pneumoniae; Excision repair; Uvr-like gene
Summary Although deficient in photoreactivation and some SOS-like functions, Streptococcus pneumoniae has the capacity to carry out excision repair when exposed to U V light. The repair ability and sensitivity to UV irradiation or treatment with chemical agents in the wild type and a UV-sensitive mutant strain indicate that UV-induced pyrimidine dimers might be repaired in pneumococcus by a system similar to the uvr-dependent system in Escherichia coll. A gene complementing the mutation conferring UV sensitivity of the mutant strain has been cloned. The coding region directs the synthesis of a polypeptide with a molecular weight of 78 kDa. The relationship with uvr-like protein in E. coli is discussed.
The bacterial response to D N A damage caused by UV light, ionizing radiations and chemical agents involves the repair of D N A by several processes (Sancar and Sancar, 1988). It has been reported that Streptococcus pneumoniae is deficient in photoreactivation (Goodgal et al., 1957) and some SOS-like functions (Gasc et al., 1980). We have used a rapid assay that we have developed in our laboratory and which allows detection of pyrimidine dimer excision in a plasmid in vivo after UV irradiation and post-incubation of the host cells. We have shown that S. pneumoniae has
Correspondence: Dr. N. Sicard, Centre de Recherche de Biochimie et de G~n&ique Cellulaires du CNRS, 118 route de Narbonne, 31062 Toulouse Cedex (France).
the capacity to carry out excision repair after exposure to UV light (Estevenon and Sicard, 1989). Excision repair can be achieved by a specific D N A glycosylase and AP endonuclease as observed in Micrococcus luteus (Carrier and Setlow, 1970; Haseltine et al., 1980) or by the uvrABC excinuclease pathway as observed in Escherichia coli (Seeberg et al., 1976; Yeung et al., 1983; Sancar and Rupp, 1983) and also in M. luteus (Tao et al., 1987; Shiota and Nakayama, 1988, 1989). We have studied the mechanism of pyrimidine dimer excision repair in S. pneumoniae by the analysis of the repair ability and sensitivity to UV irradiation and chemical agents in the wild type and a UV-sensitive mutant strain. We have cloned the gene which complements the mutation conferring radiosensitivity to this mutant strain and identified the encoded protein.
0921-8777/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
196 Material and methods
Bacterial strains and plasmid S. pneumoniae R800 was used as the parental strain (Lefrvre et al., 1979). Strain R402 is a UV-sensitive mutant, isolated after transfer of the UV-sensitive mutation 402 (Tiraby and Sicard, 1973) into R800. Cultures of S. pneumoniae were grown at 37 ° C in complete medium (Prats et al., 1985). Plasmid pSP2 was used as the cloning vector in pneumococcus (Prats et al., 1985). Determination of excision-repair capability The method has been previously described (Estevenon and Sicard, 1989). Chemicals cis-Diamminedichloroplatinum (cis-platinum) and 4-nitroquinoline-l-oxide (4NQO) were obtained from Dr. B. Salles (CNRS, Toulouse) and Dr. J. Alonso (Max Planck Institute, Berlin) respectively. Mitomycin C was purchased from Sigma Chemical Company. Restriction enzymes Restriction endonucleases and T4 D N A ligase were obtained either from Bethesda Research Laboratory Inc. (Gaithersburg, MD) or New England Biolabs, Inc. (Beverly, MA) and used as specified by the supplier. UV endonuclease from M. luteus was purchased from Applied Genetics Inc. (Freeport, NY). Survival experiments after irradiation and chemical treatments Exponentially growing cells (5 × 1 0 7 cells/ml) were harvested, washed and resuspended in the mineral buffer of the synthetic medium (Sicard, 1964). Samples of 5 ml were irradiated with UV from a G 1518 Sylvania germicidal lamp producing an incident dose rate of 2 j / m 2 / s . After irradiation, the cell suspensions were diluted and plated on agar medium. Cell survival was determined after overnight incubation at 37 o C. Samples of 5 ml were treated with mutagens as follows: after addition of chemical agents at various concentrations, the samples were kept in the dark for 1 h at 30 o C for mitomycin C and 4NQO,
and 1.5 h for cis-platinum assays. Cell survival was estimated as described above.
DNA isolation and manipulation Total D N A was isolated by the procedure of Lefrvre et al. (1979). Large-scale preparation of pSP2 plasmid was carried out according to IshHorowicz and Burke (1981) except that pneumococcal cells were lysed with a mixture of desoxycholate and sodium dodecyl sulfate (Lefrvre et al., 1979). Plasmid D N A was further purified by cesium chloride ethidium bromide density gradient centrifugation. Products from restriction digestions were analyzed on agarose gels and recovered from the gel by electroelution. D N A transformations were performed as described by Lef~vre et al. (1979). Analysis of plasmid-encoded proteins Purified recombinant plasmid D N A s were transcribed and translated according to the method of Zubay (1973). The products of the synthesis were analyzed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) (Laemmli, 1970). Results and discussion
Sensitivity to UV rays and repair capability of R800 and R402 The survival curves after irradiation of the wild-type strain R800 and its UV-sensitive derivative R402 are shown in Fig. 1. R402 exhibits a high sensitivity to UV rays. To test the repair capability of the mutant strain R402, we used a rapid assay developed in our laboratory (Estevenon and Sicard, 1989) providing an estimate of pyrimidine dimer excision repair in vivo in bacterial strains. It is based on the fact that UV endonuclease converts covalently closed circular (CCC) D N A containing pyrimidine dimers to the open circular (OC) form. Therefore pyrimidine dimer excision repair was revealed by the appearance of CCC plasmid D N A which was no longer sensitive to UV endonuclease. For the assay wild-type and mutant strains were transformed with pSP2 plasmid. The cells were exposed to UV light and incubated for 1 h in complete medium for recovery. Plasmid D N A was isolated,
197
treated with UV endonuclease and analyzed by agarose gel electrophoresis. In the wild-type strain, when the D N A was examined immediately after irradiation, all the D N A was converted to the OC form by UV endonuclease, for an incident dose of 20 or 40 J / m 2 (Fig. 2A, lanes 4 and 8, respectively). However, when post-incubation of 1 h was allowed after irradiation, a small fraction of the CCC D N A was resistant to UV endonuclease showing that some repair had taken place (Fig. 2A, lanes 6 and 10). By contrast, in the mutant strain there was no CCC D N A which was insensitive to UV endonuclease after post-incubation (Fig. 2B, lanes 6 and 10). This shows the absence of excision-repair capacity in the mutant strain R402.
10
al
¢. Q Q.
0.01
I
0
,
5
i"
q
10
20
4'0 tlV
( j/r.2 )
Fig. 1. Effect of UV rays on survival of Streptococcus pneumoniae. • R800 (wild type); o R402 (UV-sensitive mutant); * R402 (pSP800).
_
+,
Sensitivity to chemical agents Pyrimidine dimers might be removed by different incision mechanisms (Sancar and Sancar, 1988). In M. luteus the D N A glycosylase involves the specific recognition of the pyrimidine dimer (Haseltine et al., 1980) whereas the uvrABC excinuclease system in E. coil removes, through nucleotide excision repair, the U V - i n d u c e d pyrimidine dimers as well as the adducts produced
_,
_
OC
OC
CCC
CCC
A
2
3
4
5
6
7
8
9
B
Fig. 2. Incision of damaged pSP2 D N A extracted from UV-irradiated S. pneumoniae R800 (A) and R402 (B). The plasmid pSP2 has been either digested ( + ) or not digested ( - ) with UV endonuclease from M. luteus. Lanes 1-2: plasmid extracted from unirradiated bacteria. Lanes 3-4: irradiated 20 J / m 2 (A), 10 J / m 2 (B); no post-incubation. Lanes 5-6: irradiated 20 J / m 2 (A), 10 J / m 2 (B); post-incubated 1 h. Lanes 7 - 8 : irradiated 40 J / m 2 (A), 20 J / m 2 (B); no post-incubation. Lanes 9-10: irradiated 40 J / m 2 (A), 20 J / m 2 (B); post-incubated 1 h.
198 100,
.,..I
10-..,
1¢ tU O: tU 0,.
o 0,1
o
0,1
o',2
MI TOMYCINE
o',3
C (Hg/ml)
o go ~oo
2bo
4- NQO
~oo
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