Mutation induction and mutation frequency decline in halogen light-irradiated Escherichia coli K-12 AB1157 strains

Mutation Research 390 Ž1997. 85–92

Mutation induction and mutation frequency decline in halogen light-irradiated Escherichia coli K-12 AB1157 strains Anna Wojcik, Celina Janion ´

)

Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5A, 02-106 Warszawa, Poland ´ Received 30 July 1996; revised 15 November 1996; accepted 26 November 1996

Abstract The effects of halogen light irradiation on reversion of argE3 ™ Argq in E. coli K12 strain AB1157 and its mfdy mutant, and on mutation frequency decline ŽMFD. after transiently incubating irradiated bacteria under non-growing conditions were studied. The induction of mutations, the mutational specificity, and the MFD effect had the same characteristic features as those seen in E. coli B strains after irradiation with 254 nm UV light. MFD which is due to repair of premutagenic lesion in the transcribed strand of glnU gene and prevents mutations leading to supB formation, was not observed in halogen light-induced mutations in the mfd-1 strain. Overproduction of UmuDX C proteins led to a large increase in mutation frequency, which was much greater in mfdy than in mfdq strains. In bacteria irradiated with halogen light and incubated immediately in a rich medium to express mutations, the formation of supB predominated strongly over that of supEŽochre. in mfdy cells but was at a similar level in mfdq cells. Introduction of zcf117::Tn10 to AB1157 strain makes cells more sensitive to halogen light irradiation, whereas introduction of mfd-1 does not. Keywords: Halogen light-induced mutagenesis; Mutation frequency decline; E. coli; Transcription repair coupling

1. Introduction Halogen lamps are potent sources of mutations and cancers, due to emission of a broad spectrum of far and near UV radiation including UVC Ž190–280 nm., UVB Ž280–320 nm., and UVA Ž320–400 nm. w1–3x. These harmful effects may be prevented by a glass or plastic cover which cuts off UV wavelenghts below 400 nm from visible light w1–3x. The sensitivity to halogen light irradiation of E. coli strains defective in DNA-repair resembles that of bacteria irradiated with UV at 254 nm, pointing to pyrimidine ) Corresponding author: Tel.: Ž48. 658-47-66; Fax: Ž48. 39 12 16 23; E-mail: [email protected]

dimers and 4-6 pyrimidine photoproducts as responsible for the biological effects. However, the biological effects Žsurvival, mutations. of halogen light radiation are about 600-fold weaker than those of UVC-radiation in the 230–335 nm wavelength region, and longer periods of irradiation Žminutes vs second. with halogen light than with 254 nm UV are therefore required to obtain a similar biological effect w1x. This is due to the emission of a broad spectrum of UV as well as visible light by halogen lamps and to the greater conversion of photoproducts to free pyrimidines by the effect of UVA-, UVB-, and visible light on enzymatic processes of DNA photolyase w4,5x. It has been known for decades that the UV-in-

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duced mutations may undergo the phenomenon termed mutation frequency decline ŽMFD. w6–8x, when UV-irradiated bacteria are immediately and transiently cultivated under conditions which do not allow protein synthesis, certain mutations are rapidly and irreversibly lost. Recently MFD is regarded as a special case of preferential repair of premutagenic lesions in the transcribed strand of DNA which requires transcription repair coupling factor ŽTRCF. encoded by mfd, identified by Selby and Sancar w9x, and UvrABC-endonuclease system which excises a variety of modified nucleotides. In bacteria defective in either of these two genes, the MFD effect does not occur w10–13x. MFD occurs in E. coli B not only after UV irradiation, but also after exposure to EMS and in E. coli K-12 after exposure to MMS w14,15x. Here we examined mutations and MFD following halogen lamp irradiation of E. coli K-12 AB1157 strains by measuring reversions of argE3 ™ Argq. Strain AB1157 bears the argE3Žochre. , hisG4Žochre. and supE44-encoding supE amber suppressor. The vast majority of argE3 reversions occur by tRNA supressor formations due to supB, derived from one of the two glnU genes encoding tRNA for glutamine reading the codon CAA, and to supEŽochre. , derived from supE44 amber suppressor reading UAG Ž supE44 was created from glnV reading CAG.. Both mutations lead to suppressor tRNA formation resulting from GC to AT transitions. The specificity of mutations induced by halogen light and 254-nm UV radiation is therefore the same, and this system resembles that widely investigated in E. coli B strains where reversions of ochre mutations from auxotrophy to prototrophy after UV irradiation result in supB and supE ochre suppressors formation w16,17,11x.

2. Materials and methods 2.1. Bacterial strains The strains used were E. coli K-12: AB1157 with relevant genotype, argE3Žochre. , hisG4 Žochre. , supE44-encoding amber suppressor w18x, and its d e riv a tiv e s: E C 2 4 1 3 , a s A B 1 1 5 7 b u t

D umuDC595::cat w15x, EC2423, as AB1157 but zcf117::Tn10 Tet r mfd-1. EC2423 and AB1157zcf117::Tn10 Tet r were obtained in this laboratory Žby E. Gresziuk and A. Fabisiewicz. by P1-mediated transduction with NR10121 or NR10125, as donor strain. Transductants were screened for tetracycline resistance and tested for their MFD character after UV and halogen light irradiations. The mfd-1 was isolated by Witkin w7x. Construct D umuDC595::cat derived from Woodgate w19x. AB1157rpGW2123, E C 2423r pG W 2123 and A B 1157zcf117::Tn10rpGW2123 denote strains transformed with pGW2123 by routine methods for plasmid transformation w20x; Plasmid pGW2123 Ž umuDX C Ap r . was from Dr. G.C. Walker w21x; NR10121 and NR10125 from the collection of Dr. R.N. Schaaper w22x. 2.2. Media Bacteria were grown in Luria broth w23x, and suspended in C salts w24x with 0.5% of glucose for halogen light irradiation. Tetracycline Ž15 mgrml. was added to grow zcf117::Tn10 containing bacteria. -Arg, and -His plates, contained minimal media enriched with O.4% glucose and all the requirements ŽThr, Leu, Pro, Thi. except Arg, or His, respectively. 2.3. Halogen lamp irradiation Bacteria grown in LB to about 2–3 = 10 8 cellsrml were centrifuged, resuspended in C-salts with glucose and 9 ml samples in petri dishes Ž90 mm. were irradiated with 150-W halogen lamp ŽPolamp. placed 20 cm above the plate. One minute of irradiation emitted Žin Jrm2 .: UVC, 162; UVA, 846; UVB, 732, measured using an IL-1500 radiometer ŽDexter Industrial Green Newburyport USA. and the appropriate probe. After the indicated period of irradiation the bacteria were diluted into LB Ž0.3 ml ™ 3 ml. either immediately, or after 20 min of incubation in C-salts with glucose Žconditions for MFD., and then grown overnight before viable counts and Argq revertants were scored. Before and after irradiation the number of cells Žsurvival. was estimated. Incubation was at 378C and irradiated bacteria were kept under dim yellow light or in darkness.

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2.4. Analysis of Arg q reÕertants The Argq revertants, selected on -Arg plates, were first examined for their requirement for His Žby plating on -His and -Arg plates. and then the ArgqrHisy isolates Ž90–100% of all the halogen light-induced Argq revertants. were tested for suppressor activity. The ArgqrHisy phenotype can be caused either by a back mutation at the argE3 locus Žmutational target T-T., by supB suppressor formation Žmutational target T-C in the transcribed strand. or by conversion of supEŽamber. to supEŽochre. Žtargeted at T-C located in the coding strand.. To discriminate between these mechanisms, 10–35 independent revertants were isolated and their abilities to support growth of amber ŽB17. and ochre Žoc427, ps292, ps205. mutant T4 phages was determined. A pattern of phage growth, respectively, of Žq y y y. indicates a back mutation and Žq q q q. or Žy q q q. patterns indicate supB, or supEŽochre. suppressor mutations w25,26x. 3. Results 3.1. Halogen light-induced argE3 ™ Arg q reÕersion The dose dependency of the frequency of halogen light-induced argE3-Argq reversion in two bacterial strains which differ in TRCF activity ŽAB1157 mfdq and, EC2123 mfdy. is shown in Fig. 1A. In the mfdq strain the mutation frequency increased with time of irradiation and reached a plateau after 5 min, whereas in mfdy strain, the mutation frequency was higher than in mfdq until 4 min of irradiation Žwith a 2.5-fold higher peak after 3 min. and then reached a plateau level similar to that in the mfdq strain. This was in contrast to the effect of UV radiation, where the mutation frequency was about 5-fold higher in mfdy than in mfdq bacteria, independently on the UV fluence w22,7x. The reasons for this difference may be complex. First, there is more time for excision repair during irradiation with halogen light for several minutes than during irradiation with UV254 nm for several seconds. Second, the visible light emitted by a halogen lamp together with photolyase activity may convert photoproducts to monomers or hasten their exci-

Fig. 1. ŽA. Mutation frequency based on reversion of argE3™ Argq in AB1157 mfdq Ž'. and in the EC2423 mfd-1 derivative Žv .. ŽB. Survival of AB1157 Ž'., EC2423 Žv . and AB1157zcf117::Tn10 ŽB. after halogen light irradiation. 1 minute of irradiation corresponded to an UVC emission of 162 Jrm2 . The data are means of 3–4 experiments, each made in duplicate; the SD did not exceed 42%.

sion mediated by UvrABC-endonuclease w5,27x, and the efficiency of these processes may be different in the mfdy and mfdq strains. Alternatively, since halogen light is a weaker inducer of the SOS response than UV a shortage of UmuDC proteins is a limiting factor of mutational events in EC2423 mfdy strain. The SOS proteins are necessary for expression of mutations induced by UV254 w28,29x. It should be stressed that defect in the mfd gene does not affect the sensitivity of bacteria to halogen light irradiation. Note that introduction of the zcf117::Tn10 transposon itself makes bacteria more sensitive to halogen light irradiation to the same extent as introduction of the zcf117::Tn10 mfd-1 ŽFig. 1B.. Moreover, introduction of zcf ::Tn10 into

A. Wojcik, C. Janionr Mutation Research 390 (1997) 85–92 ´

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AB1157 makes bacteria more resistant to halogen light-induced mutagenesis Žsee Table 1.. The reason for that, why introduction of the Tn10 transposon make bacteria more sensitive to halogen light-irradiation but more resistant to halogen light-induced mutagenesis, is not clear. 3.2. The MFD effect in halogen light-irradiated bacteria When bacteria were irradiated with halogen light and then exposed to condition for MFD Žincubation in C salts with glucose. before mutations were expressed, the mutation frequency declined in mfdq bacteria whereas in mfdy bacteria it showed little or no decrease ŽFig. 2.. Analysis of the mechanism of

Argq reversions revealed that in mfdq strain this resulted from supB and supEŽochre. suppressor formations at the ratio of approximately G 1:1 but after MFD this ratio was from 1:3 till 1:20. The decline in mutation frequency was therefore greatly due to a decline in reversion arising by supB suppressor formation ŽFig. 3A and Fig. 4.. In contrast in mfdy bacteria reversions to Argq were due predominantly or exclusively to supB formation before or after incubation under conditions for MFD. A longer time of irradiation or incubation under conditions of MFD clearly favoured supB formation in the mfdy strain ŽFig. 3BFig. 4.. In the mfdy strain, lesions leading to supB were therefore, either not repaired andror lesions leading to supEŽochre. formation were repaired preferentially.

Table 1 Survival, frequency of reversions and MFD effect after halogen light irradiation in the indicated bacterial strains Irradiation time

Survival

Frequency of reversion ŽArgq revertantsr10 8 cells. MFD starvation time Žmin. effect

Žmin.

Ž%.

0

EC2413 Ž D umuDC .

0 2 5

100 23.4 " 8.2 1.29 " 0.32

AB1157

0 2 5

100 70.3 " 10.5 51.8 " 5.0

2.3 " 0.8 643 " 148 1676 " 321

– 172 " 35 418 " 124

– 73.2 75.1

AB1157r pGW2123

0 2 5

100 62 31.5 " 5.9

22.8 " 11.6 1042 2400 " 397

– 486 1015 " 350

– 53.4 57.7

AB1157 but zfc117::Tn10

0 2 5

100 30.9 " 9.9 5.5 " 2.1

3.0 " 1.1 471 " 96 693 " 337

–

– 79.0 81.2

AB1157 but zcf117::Tn10 rpGW2123

0 2 5

100 73.4 26.2

24.6 3300 3451

– 725 1452

– 78.0 57.9

EC2423 Ž mfdy .

0 2 5

100 29.8 " 8.1 6.9 " 1.7

3.8 " 1.6 1631 " 464 1595 " 393

– 1595 " 295 1493 " 90

– 2.2 6.4

EC2423 Ž mfdy .r pGW2123

0 2 5

100 54.4 " 13.2 17.9 " 2.1

25.2 " 6.9 5230 " 648 10458

– 4595 " 928 7956

– 12.1 23.9

Strain

20 1.4 3.2 1.7

Ž%.

– 4.3 3.0

99 " 110 130 " 50

– – –

frequency of reversions in starved bacteria x100. frequency of reversion in non y starved bacteria The data are means of 2–4 experiments, each made in duplicate. Data which were results of 2 experiments have no "SD values. )

MFD is calculated as 100 y

)

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Fig. 2. Mutation frequency decline after irradiation of mfdq ŽAB1157. or mfdy ŽEC2423. bacteria for the indicated time and further incubation for 20 minutes in C-salts with 0.5% glucose. The data show the frequency of reversion to Argq in starved cells as the percentage of that in non-starved cells.

3.3. MFD in bacteria transformed with pGW2123 The SOS proteins: RecA, UmuD Žor more precisely its processed form UmuDX ., and UmuC play central roles in UV-induced mutagenesis w29–33x. In bacteria EC2413 strain with a deleted umuDC operon halogen light-induced Žlike UV254 nm -induced. mutagenesis does not occur ŽTable 1, first position.. We therefore transformed strains AB1157 and EC2423 with the plasmid pGW2123 which bears umuDX C operon and examined the Argq reversion frequency induced by halogen light irradiation and the MFD effect. The data from these experiments ŽTable 1 and Fig. 5. lead to a number of conclusions. First in the absence of TRCF protein Ž mfdy strain. overproduction of UmuDX C proteins had a much more pronounced effect on the induction of Argq revertants, than in the presence of TRCF Ž mfdq strain.. Second, the frequency of induced Argq reversion in the mfdy strain exceeded that seen in mfdq at all irradiance values. Third, the MFD effect occurs in the mfdq strain in a similar manner to that seen in non-transformed bacteria. Fourth ŽFig. 5. the Argq revertants in mfdy strain which resulted from supB suppressors formation predominated over those which resulted from supEŽochre. formation, whereas in mfdq

Fig. 3. ŽA,B. Frequency of reversion to Argq arising by supB Ž'. or supEŽochre. ŽB. suppressor formation in AB1157 mfdq ŽA. and in EC2423 mfdy ŽB. after the indicated period of irradiation. Bacteria non-subdued to MFD, continuous line; subdued to MFD, dashed line. The data were calculated from the ratio of supB to supEŽochre. activity in samples of 10–35 Argq revertants. Each point represents the mean of 2–4 experiments, each made in duplicate.

Fig. 4. Distribution Žin percentage. of supB ŽB. and supEŽochre. ŽI. among the ArgqrHisy revertants of AB1157 Ž mfdq . and EC2423 Ž mfdy . strains. Bacteria were irradiated for 2 min Ž1,2. or for 5 min Ž3,4. and incubated to express mutation either immediately Ž1,3. or after 20 min of starvation Ž2,4..

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Fig. 5. Distribution of of supB ŽB. and supEŽochre. ŽI. among the ArgqrHisy revertants of AB1157rpGW2123 Ž mfdq . and EC2423rpGW2123 Ž mfdy . strains. For further details see the legend under Fig. 4.

the number of Argq revertants bearing supB or supEŽochre. suppressor was similar. This is consistent with the notion that there are much more premutagenic lesions than UmuDX and UmuC proteins required for their mutagenic processing both in the mfdq and specially in mfdy strains. Therefore either the Mfd protein competes with UmuDC for the damaged sites, or the repair of of premutagenic lesions leading to supB formation occurs so efficiently in mfdq strain that the majority of them are already repaired at zero time of starvation. The results on AB1157-zcf ::Tn 10 and its pGW2123 transformant show that Ži. induction of reversions was lower in AB1157-zcf::Tn10 than in AB1157 Žii. reversions were mainly due to supB and supEŽochre. formations Žiii. transformation of AB1157-zcf::TN10 with pGW2123 increases Argq reversions to the level of close to that observed in AB1157_ pGW2123 transformants ŽTable 1.. Transformation with pGW2123 resulted in a uniform sensitivity of bacteria to halogen lamp irradiation by rendering AB1157 cells more sensitive and EC2423 more resistant.

4. Discussion 4.1. Mechanism of halogen light-induced mutations There is no doubt that halogen light emitted directly from the uncovered source is a serious risk for

organisms; the most harmful component are the UV 254 nm and fluorescent radiations which contribute to DNA damage, mutations, malignant transformations, skin cancer and genotoxicity Žw1x, and cited therein.. In this paper the mutagenicity of halogen light for bacteria was studied with a special reference to preferential DNA repair and the phenomenon of MFD. The mutagenic targets in DNA for UV damage were found to be Ži. 5X- TCAA-3X in the transcribed strand of glu-tRNA gene causing supB formation, and Žii. 3X-ATCT-5X in the coding strand of supE44-encoding amber tRNA suppressor, causing supEŽochre. formation Žthe targeted bases are underlined.. In both cases C residues are a part of anticodon tRNAs Žitalic. and are flanked by T, and the mutational pathway involves a GC to AT transition; and Žiii. ATT – encoding an ochre triplet situated on the transcribed strand at argE3 ochre site, leading to back mutations by AT transversions or UAG codon formation Žsuppressed by supE44 . AT to GC transition. Therefore in our experimental system Žand in WU36 of E. coli B strains. the phenomenon of MFD, being a reflection of preferential repair of DNA, is observed only as a decline in mutation frequency leading to supB suppressor formation. It is noteworthy that after halogen light irradiation, back mutations were observed only in the uÕrAy background Ždata not shown.. This suggests that T-T photoproducts at this site are efficiently removed by UvrABC-endonuclease, independently of whether the bacteria are mfdy or mfdq. 4.2. Role of Mfd protein and effect of oÕerproduction of UmuDX C proteins The role of MFD is ‘displacement of the stalled RNA polymerase from transcribed strand of DNA and the recruitment of repair enzyme to the damage site’ w34x. The MFD protein possesses UvrA and RNA polymerase binding sites, and helicase motifs with sequences of identity to RecG protein, but the helicase substrate is unknown. Preferential strandspecific re-pair assayed in Õitro can be inhibited by an excess of UvrA which binds to free Mfd w13x. To repair lesions, most of the repair enzymes require the double-stranded structure so that ‘transcription bubbles’ therefore should be closed. RecA, UmuDX2UmuC and DNA polymerase III is required

A. Wojcik, C. Janionr Mutation Research 390 (1997) 85–92 ´

to bypass of uninformative, photoproduct containing, site in replicated DNA w28,33x. The Mfd and UmuDC proteins are therefore engaged in two activities, transcription ŽMfd. and replication ŽUmuDX C.. Overproduction of UmuDX C from plasmid pGW2123 leads to a much greater increase in halogen light-induced argE3 ™ Argq reversions in mfdy strain EC2423 than in the mfdq AB1157 ŽTable 1.. This may indicate that the Mfd and UmuDX C proteins either compete for the lesions induced by halogen light andror, since mfdy mutations do not reduce survival but only influence the pattern of repair, that the much higher supB suppressor formations in mfdy than in mfdq bacteria is merely a reflection of the loss of preferential DNA repair. We have no grounds for explaining why the introduction of zcf::Tn10 renders bacteria more sensitive to halogen light. The effect, however, is more general and we have observed sensitization of bacteria to halogen, and UV irradiation, after introducing the following constructs into bacteria: uÕrA::Tn10 Ž Õs uÕrA6 . and leu::Tn10.

w6x

w7x w8x

w9x

w10x w11x

w12x

w13x

w14x

Acknowledgements w15x

We are grateful to Drs. R. Woodgate and I. Fijalkowska for bacterial strains, G.C. Walker for pGW2123 plasmid and to Dr R. Hancock for language correction. This work was partially supported by The State Committee for Scientific Reasearch, Grant No G31.

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