Inducible stable DNA replication in Escherichia coli uvr− and uvr− cells treated with genotoxic chemicals

Mutation Research, 281 (1992) 63-66 © 1992 Elsevier Science Publishers B.V. All rights reserved 0165-7992/92/$05.00

63

MUTLET 00610

Inducible stable DNA replication in

E s c h e r i c h i a coli u v r +

and

uvr-

cells,

treated with genotoxic chemicals Franti ek Magek and Milena Sedliakov Department of Molecular Genetics, Cancer Research Institute, Slovak Academy of Sciences, Bratislava (Czechoslovakia) (Received 24 November 1990) (Revision received 23 August 1991) (Accepted 18 September 1991)

Keywords: Stable DNA replication; Induction; Genotoxic chemicals; Uvr defective; Escherichia coli

Summary Inducible stable DNA replication (iSDR) provoked by a damaging treatment with MMS, MNU, MNNG, NFAA, NFN, 4NQO, NAL or MMC, was followed in both repair-competent E. coli PQ35 and its uvrA derivative E. coli PQ37. In contrast to SOS-inducible mutagenesis, which is more pronounced in excision-deficient cells, iSDR was more obvious in repair-competent cells. This may be due to special features of iSDR and need not indicate involvement of the uvrA gene product in it.

DNA replication of undamaged E. coli originates from a unique site of the chromosome, designated as oriC. This step requires several DNA replication initiating proteins. When cells are incubated in the presence of chloramphenicol, DNA synthesis is gradually inhibited since the ongoing cycles are terminated and new cycles cannot be initiated (Hanawalt et al., 1961; Magee and Kogoma, 1990). In contrast, in cells transiently exposed to thymidine starvation or nalidixic acid treatment, continuing DNA synthesis has been observed, despite the presence of chloramphenicol. This type of DNA synthesis was considered termination-defective and called stable DNA replication (Kogoma and Larke, 1970).

Correspondence: Dr. F. Ma~ek, Department of Molecular Genetics, Cancer Research Institute, Slovak Academy of Sciences, Mlynsk~ Nivy 59, 812 32 Bratislava (Czechoslovakia).

Later a similar mode of DNA replication was observed after UV irradiation or after treatment with mitomycin C, when protein synthesis was allowed to take place between the damaging treatment and the chloramphenicol treatment. It has been concluded that this type of replication is one of the SOS damage-inducible responses of cells (Kogoma et al., 1979) and was designated iSDR (inducible stable DNA replication). iSDR is a special type of DNA replication since it does not require concomitant protein and RNA syntheses and originates from at least 2 sites of DNA chromosome different from oriC (Magee and Kogoma, 1989). This replication becomes evident after a period of inhibition of protein synthesis during which the ongoing replication cycles are terminated. Its regulation is not clear. iSDR was found to be dependent on recBC but independent of uvrA (Kogoma et al., 1979;

64 Khidhir et al., 1985). However, in excision-proficient cells (unlike excision-deficient ones) an increase in the rate of iSDR has been observed 60 min after UV irradiation which might suggest involvement of DNA repair (Kogoma et al., 1979). Here we compare the kinetics of iSDR in the strain of E. coli uc'r + and its uvr- derivative after various damaging treatments. Materials and methods

Bacterial strains and culture conditions. Escherichia coli PQ35 (uvr +) and Escherichia coli PQ37 (uvr-) have been described by Quillardet and Hofnung (1985). Both carry the rfa mutation, which allows better diffusion of chemicals into the cell. The culture medium consisted of Bacto tryptone 10 g, Bacto yeast extract 5 g, NaC1 10 g/1 of distilled water and 20 p.g/ml of ampicillin. Assay of stable DNA repfication The cells were grown until the optical density reached 0.20 (at 600 nm). Chloramphenicol (Cam, 40/xg/ml) was immediately added to a portion of the culture and incubated for 180 rain. To the other portion(s) a chemical to be tested was added in a given concentration and cells were incubated for 90 min. After harvesting by centrifugation and resuspending in fresh medium, the suspensions were adjusted to optical density 0.20; Cam (40 Ixg/ml) was added and suspensions were incubated for 180 min. Then [6-3H]thymidine was added and cells were incubated for 120 min. At the given time 0.1 ml of bacterial suspension was taken, applied to a Whatman 3 paper disk, washed with 5% trichloroacetic acid and ethanol and counted in a Beckman 1801 liquid scintillation counter.

(Milwaukee, WI, U.S.A.); 3-(5-nitro-2-furyl) acrylic acid (NFAA) and 5-nitro-2-furfurylnitrate (NFN) were gifts of Dr. Krutogikovfi (Slovak Technical College, Bratislava, Czechoslovakia); ampicillin was from Spofa (Prague, Czechoslovakia); deoxyadenosine from Calbiochem (San Diego, CA, U.S.A.); Bacto tryptone and Bacto yeast extract from Difco (Detroit, MI, U.S.A.). [6-3H]Thymidine (3H-Tdr) was from the Institute for Research Production and Uses of Radioisotopes (Prague, Czechoslovakia). Results

In our experiments the following genotoxic chemicals were used: alkylating agents, namely MMS, MNU, MNNG, acting predominantly through methylation of 0 6 , N 7 guanine and N 3 adenine (Walker, 1984); nitrofurans, namely NFAA, NFN, producing single-strand breaks of DNA (McCalla et al., 1971); 4NQO, acting through the formation of 4NQO-guanine and 4NQO-adenine adducts (Ikenaga et al., 1975), NAL, inhibiting DNA replication by inhibiting subunit A of DNA gyrase (Gellert et al., 1977) and forming a complex with DNA and gyrase that produces double-strand breaks of the DNA (Snyder and Drlica, 1979; Morrison et al., 1980) as well as MMC, making cross-links between two DNA strands thus blocking DNA replication and transcription (Sassanfar and Roberts, 1990). As shown in Fig. 1 all these chemicals induced some DNA replication in both types of the E. coli strains, which indicated that it was a regular response to DNA damage. The kinetics of 3H-Tdr incorporation was always higher in E. coli PQ35 (uvr +) cells (Fig. 1 left) than in the uvr- derivative (Fig. 1 right). Discussion

Chemicals 4-Nitroquinoline 1-oxide (4NQO) was purchased from Sigma (St. Louis, MO, U.S.A.); nalidixic acid (NAL), mitomycin C (MMC), 1methyl-3-nitro-l-nitrosoguanidine (MNNG) and chloramphenicol (Cam) from Serva (Heidelberg, Germany); N-methyl-N-nitrosourea (MNU) from ICN Biochemicals (Cleveland, OH, U.S.A.); methyl methanesulfonate (MMS) from Aldrich

Generally, SOS responses are more visible in excision-deficient cells than in excision-proficient ones where lesions triggering the SOS signal are quickly removed from the DNA and SOS induction is faster switched off (Radman, 1975; Radman et al., 1976). However, our data (Fig. 1) indicate that this rule does not hold for iSDR, since the incorporation of 3H-Tdr was always

65

PO 35

PQ 37 I

7 6 5

~ S_.t.~

4

MNNG 20 I ~_-e MMS 2

~_

MNU 400

3

MNNG jo."~.---o

2 ___.o~O o;,--0---"9 , ,

,¢-~"~?---Y

MMS

T

7 6 NFN 20 NFAA 10

×) /

I ~f..Q~ I C ~ L - - I o---', 7

6

e/

•

J

45 T-~'""4~ /e" . ~

~ N cF--v NAL 40

I MMC1 I

"

30 60 g0 120 Fig. 1. iSDR of

E. coil

j . . . - o NFAA N , , x

O4N00 25

3= 2

F

4NOO ~

MMC"

30 60 g0 120 MINUTES

PO35 (uvr + ) and PO37 (re'r-) after 3

h incubation with chloramphenicol. The most effective concentrations of chemicals (selected in preliminary experiments; not shown) were applied. The chemicals were added in exponential growth phase and the cells incubated for 90 min. Cells were transferred into fresh cold medium containing cbloramphenicol and incubated for 3 h. Then [6-~H]thymidine and deoxyadenosine were added and cells were incubated for another 2 h. During this period samples were taken at indicated intervals.

greater into the DNA of excision-proficient cells than into that of the uLTA mutants. The question, however, arises whether the 3H incorporation observed in excision-proficient cells actually reflects stable DNA replication. As is well known, initiation of new replication cycles cannot take place in chloramphenicol. Since in our conditions cells were incubated for 3 h with c h l o r a m p h e n i c o l before the 3H-Tdr was added, the possibility that its i n c o r p o r a t i o n reflects the c o n t i n u a t i o n of cycles initiated prior to the addition of c h l o r a m p h e n i c o l is unlikely. A n o t h e r possibility, n a m e l y that the 3 H - T d r i n c o r p o r a t i o n

into the DNA of excision-proficient cells represents repair synthesis needed for filling the gaps after excised lesions, cannot be excluded. However, in UV-irradiated E. coli excision repair terminates within 120 min in dependence on UV dose (Setlow, 1967; Sedliakov~ et al., 1971; Magek a n d Sedliakovfi, 1977). Starting from this we favor an a s s u m p t i o n that the kinetics of stable D N A replication is g r e a t e r in excision-proficient cells t h a n in excision-deficient ones. However, it may be due to some special features of i S D R a n d n e e d not indicate i n v o l v e m e n t of the u t , r A gene p r o d u c t in this process. It has b e e n f o u n d that the R e c A p r o t e i n is the only din p r o t e i n that must be p r e s e n t in an increased level for i S D R to occur (Magee a n d Kogoma, 1989). Since this p r o t e i n is relatively stable a n d its level does not decrease in non-dividing cells (Salles and Paoletti, 1983), i S D R might c o n t i n u e in the p r e s e n c e of c h l o r a m p h e n i col even several hours after the removal of the lesions triggering the SOS signal, Because of this, i S D R might be faster in r e p a i r - c o m p e t e n t cells where the majority of lesions r e t a r d i n g D N A replication was removed from the t e m p l a t e a n d might c o n t i n u e in spite of the absence of the triggering lesions.

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66 Kogoma, T., T.A. Torrey and M.J. Connoughton (1979) Induction of UV-resistant DNA replication in Escherichia coli. Induced stable DNA replication as an SOS function, Mol. Gen. Genet., 176, 1-9. Magee, T., and T. Kogoma (1989) DNA damage-inducible origin of DNA replication in Escherichia coli, in: Trends in Comparative Molecular Genetics, FEBS Symposium, June 18-24, Liblice, Abstracts, p. 30. Magee, T.R., and T. Kogoma (1990) Requirement of RecBC enzyme and an elevated level of activated ReeA for induced stable DNA replication in Escherichia coli, J. Bacteriol., 172, 1834-1839. Ma~ek, F., and M. Sedliakov~_ (1977) Depressibility of excision repair mechanism in Escherichia coli Br Hcr + cells, Studia Biophys., 64, 1-6. McCalla, D.R., A. Reuvers and C. Kaiser (1971) Breakage of bacterial DNA by nitrofuran derivatives, Cancer Res., 31, 2184-2188. Morrison, A., N.P. Higgins and N.R. Cozzarelli (1980) Interaction between DNA gyrase and its cleavage site on DNA, J. Biol. Chem., 255, 2211 2219. Quillardet, P., and M. Hofnung (1985)The SOS chromotest, a colorimetric assay for genotoxins: procedures, Mutation Res., 147, 65-78. Radman, M. (1975) SOS hypothesis: phenomenology of an inducible DNA repair which is accompanied by mutagenesis, in: P.C. Hanawalt and R.B. Setlow (Eds.), Molecular Mechanisms for Repair of DNA, Plenum, New York, pp. 335-337.

Radman, M., P. Caillet-Fauquet, M. Defais and G. Villani (1976) The molecular mechanism of induced mutagenesis and an in vitro biochemical assay for mutagenesis, in: R. Montesano, H. Bartsch and L. Tomatis (Eds.), Screening Tests in Chemical Carcinogenesis, IARC, Lyon, pp. 537545. Salles, B., and C. Paoletti (1983) Control of UV induction of RecA protein, Proc. Natl. Acad. Sci. (U.S.A.), 80, 65-69. Sassanfar, M., and J.W. Roberts (1990) Nature of the SOS-inducing signal in Escherichia coli: the involvement of DNA replication, J. Mol. Biol., 212, 79-96. Sedliakovfi, M., F. Ma~ek and L. Bernfitovfi (1971) Relationship between survival and thymine-dimer excision in ultraviolet-irradiated Escherichia coli, Photochem. Photobiol., 14, 597-605. Setlow, R.B. (1967) Repair of DNA, in: V.V. Koningsberger and L. Bosch (Eds.), Regulation of Nucleic Acid and Protein Biosynthesis, Elsevier, Amsterdam, pp. 51-62. Snyder, M., and K. Drlica (1979) Gyrase on the bacterial chromosome, DNA cleavage induced by oxolinic acid, J. Mol. Biol, 131,287-302. Walker, G.C. (1984) Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli, Microbiol. Rev., 48, 60-93.

Communicated by J. Veleminsk9