Mutation Research, 190 (1987) 77-81 77 Elsevier

MTRL 0936

Inducible D N A polymerase I synthesis in a UV hyper-resis tant mu tan t of Escherichia coli

s.I. Ahmad a and C.A. v a n Sluis b

aDepartment of Life Sciences, Trent Polytechnic, Burton Street, Nottingham NGI 4BU (Great Britain) and aLaboratory of Molecular Genetics, State University of Leiden, P.O. Box 9505, 2300 RA Leiden (The Netherlands)

(Accepted 29 August 1986)

Keywords: DNA polymerase I, inducible, synthesis; Escherichia coli, UV hyper-resistant mutant; argH locus; Recombinant plasmids.

plasmids.

Summary

A mutant of Escherichia coli which is more resistant to shortwave UV light than its wild-type parent strain and which can synthesise DNA polymerase I constitutively has been further analysed. It carries two muta- tional alleles which are located about 1.5 min apart and cotransducible by P1 with the argHlocus. The two mutational alleles have been segregated and their analysis shows that one of them is responsible for UV hyper-resistance whereas the other mutation confers UV sensitivity.

Recombinant plasmids carrying various sections of the polA regulatory region, linked to a galK gene, were introduced into the mutant strains. Analysis of galactokinase shows that the enzyme activity in the UV hyper-resistant mutant is increased. The results suggest that the synthesis of DNA polymerase I in E. coli is inducible.

DNA polymerase I of Escherichia coli is respon- sible for catalysing the addition of deoxy- ribonucleotides in the DNA [6]. One biological role of this enzyme is repair replication occurring during DNA replication and repair of damage to DNA [4]. The synthesis of this enzyme in E. coli has been suggested as being constitutive [11]. On the linkage map of E.coli the polA gene is located at 86 min [2] and the DNA sequence of this gene has now been established [5].

Correspondence: Dr. S.I. Ahmad, Department of Life Sciences, Trent Polytechnic, Burton Street, Nottingham NG1 4BU (Great Britain).

In the course of our study on DNA damage and repair in E. coli a mutant strain (here described as UV-resistant) was isolated which showed hyper- resistance towards a number of DNA-damaging agents including short-wave UV, mitomycin C, nalidixic acid, 5-fluorouracil and thymineless death [1], Analysis of a number of enzyme ac- tivities involved in DNA repair showed that at least two of them, namely the DNA polymerase I and the endonuclease I, were present at considerably increased levels. The levels of DNA polymerase I and endonuclease I are increased 36- and 12-fold respectively [1]. It is conceivable that the increased activity of polymerase I in the mutant strain is caused by a conformational change in the protein.

0165-7992/87/$ 03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

78

Alternatively, the level of expression of the PolA operon might be increased in the mutant strain. To understand the exact reason leading to the increas- ed UV resistance, the mutant strains and various derivatives were analysed.

Materials and methods

Bacterial strains. Escherichia coil Hfr KL14 [2]; SA236, a UV-resistant derivative of KL14 [1]; CGSC4535, F - , thi-, ilv-, argH-, metB-, rha-, lacY- or ZT, gal-, str-A (from B.J. Bachmann); SA322, an arg ÷ UV-sensitive derivative of CGSC4535 by P1 transduction, donor strain: SA236; SA323, an arg + UV-resistant derivative of CGSC4535 by P1 transduction, donor strain: SA236. SA242, F - , thr-, leu-, thi-, pro-, thy-, UV-resistant, derived from a thy-derivative of ABl157 by conjugation with SA236 selecting his + recombinants.

Plasmids. Recombinant plasmid pKIM carries galK without any promotor and amp-r [5]; plasmid pCJ63 carries -99 to + 109 nucleotide sequence from the polA structural gene linked to a pro- motorless galK, amp-r; pC J93 carries -484 to + 109 nucleotide sequence from the polA struc- tural gene linked to a promotorless galK, amp-r (C. Joyce, personal communication; see also [5]).

Measurement of cell survival after UV irradiation A freshly grown culture in nutrient broth was

serially diluted and 0.1-ml aliquots from various dilutions were plated on nutrient agar plates. The cells were then exposed to a germicidal UV lamp for variable periods and colonies appearing after 24 h incubation at 37°C were recorded.

The P1 transduction. Experiment was carried out according to Miller (1972).

Transformation. Experiment was carried out as described by Maniatis et al. (1982).

Enzyme assay Galactokinase activity was determined ac-

cording to McKenney et al. (1981). ~-Lactamase activity was determined according

to O'Callaghan et al. (1972).

Results and discussion

Genetic analysis of the UV-resistant phenotype A preliminary genetic analysis of the mutant

strain, SA236, revealed that the UV-resistant phenotype can be transferred to another E. coli strain by cellular conjugation [1]. The recipient strain, SA242, receiving the mutant phenotype was as resistant to UV as the parental donor strain (data not presented). Subsequent results obtained by P1 transduction showed that the UV-resistant strain, SA236, carried at least two mutational loci; one being responsible for conferring the UV- resistance phenotype on the cell and the other resulting in an increased UV sensitivity of the strain (Fig. 1). Both the mutational loci are P1 co- transducible with the argBCH operon and are located about 1.5 min apart on the chromosome (unpublished data). These separate locations enabled the construction of strains each having one mutant allele. SA322 and SA323 are UV-sensitive and UV-resistant mutants respectively.

Expression of galK linked to the regulatory se- quence of poiA in the mutant bacteria

Plasmids, pC J63 and pC J93 carry DNA segments preceding the polA structural gene linked

1OO

_ s°l \°\o~

• \\ i 20

o.

10 SA 323. CGSC 4 5 3 5 o

SA 3 2 2 ,

5

2() (~0 100 140 180 J/m 2

Fig. 1. Bacterial kill curve of various strains of Escherichia coli by far-UV. CGSC4535 is the parental strain; SA323 is the UV hyper-resistant derivative of CGSC4535 a n d SA322 is the UV- sensitive derivative o f CGSC4535.

to a promotorless galK gene. In the fusion plasmids the expression of galactokinase is now under control of the polA regulatory region. The segment of the polA regulatory region carried in pC J63 is -99 to + 109 nucleotide sequence en- compassing one possible promotor region whereas pC J93 carries -484 to + 109 nucleotides of the same region of the genome (C.M. Joyce, personal communication).

The two plasmids were introduced by transfor- mation into wild-type E. coli KLI4 and its UV- resistant derivative, SA236. Measurement of galac- tokinase activity in these strains shows (Table 1) that the enzyme activity is significantly increased in the UV-resistant strain, SA236 in comparison with the activity in the wild-type parent strain. Further- more, there is a difference in the increased activity in strains having pC J63 from that of the strain having pC J93. Whereas with pC J93 the enzyme ac- tivity is increased 25-fold, with pC J63 the increase is 16-fold only.

Next the two plasmids were introduced into SA323 (the UV-resistant transductant) and into SA322 (the UV-sensitive transductant). Measure-

79

ment of galactokinase activity in these strains shows that the activity is increased only in strain SA323 (Table 1).

Since DNA polymerase is also involved in plasmid replication it may be that the elevated level of galactokinase activity in various strains is due to an increase in plasmid copy number rather than an increase in the expression of the operon. To clarify this point, plasmid copy number was measured in various strains by determining the levels of /~- lactamase in the cells. The results show that there is insignificant fluctuation of plasmid copy number in the strains analysed (Table 1).

From the results it is clear that the synthesis of DNA polymerase I in E. coli is inducible and we propose a model for the genetic control mechanism. We suggest that the PolA operon in E. coli is negatively controlled, analogous to the con- trol mechanism observed for the Lac operon. The regulatory region of polA contains two putative promotors, P1 and P2, and two operators, O1 and 02. These promotors and operators are affected by a repressor protein synthesised by the UVH (UV-resistant) allele located near the argH locus.

TABLE 1 INTRACELLULAR GALACTOKINASE AND ~-LACTAMASE LEVELS IN Escherichia coli DERIVATIVES HARBOURING VARIOUS polA-galK OPERON FUSION PLASMIDS The galactokinase and/3-1actarnase activities are expressed as units/108 bacterial cells.

Strains Galactokinase activity /3-Lactamase activity

pKO4/KLI4 88 N.A. pKO4/SA236 242 N.A. pKO4/CGSC4535 60 850 pKO4/SA322 208 1514 pKO4/SA323 1436 1386

pCJ63/KL14 1321 N.A. pCJ63/SA236 3954 N.A. pCJ63/CGSC4535 1248 370 pCJ63/SA322 2298 1259 pCJ63/SA323 6023 1147

pCJ93/KL 14 527 N.A. pCJ93/SA236 6244 N.A. pCJ93/CGSC4535 976 615 pCJ93/SA322 1949 1159 pCJ93/SA323 25590 2396

N.A., Data not available.

80

P2 p c j 6 3 I

PI 01 (dnaA) P2 p c j 9 3 I e ' - 1 3 2 - 1 ( ) 0

Genomic P1 Ol ( dnaA ) P2 s e l ~ n e n t I i j

Fig. 2. Schematic regulatory region of polA in E. coil

02 I

02

In the wild-type strain the UVH product binds to both operators and a basal level synthesis of DNA polymerase I ensues. We furthermore propose that both regulatory sites are present in pC J93 but not in pC J63. In pC J63 the P1 and the O1 sites are deleted (Fig. 2). In addition, pC J93 contains a 9 nucleotide sequence located between -132 and - 124 base pairs which is a binding site for DnaA protein [5]. The DnaA protein has been suggested as a regulatory molecule that binds to a 9 base pair consensus sequence (dnaA box) having a negative effect on the expression of several operons [3].

Thus in the wild-type strain of E. coli (e.g. KL14) the expression of the poiA operon is con- trolled by two kinds of repressor molecules. One of them is synthesised by the UVH allele whereas the other is DnaA protein.

Our model for the control of DNA polymerase I synthesis is supported by the observation that in the wild-type strain of E. coli, carrying pC J63, the galactokinase activity is higher than in the wild- type strain carrying pC J93 (Table 1). This is because the dnaA box in pC J63 is deleted and repression imposed by the DnaA box protein is eliminated. Consequently, in this case the operon is expressed only from the P2 promotor without control from DnaA protein. In the UV-resistant strains the putative UVH gene product is either in- active or has reduced activity, thus both the pro- motors P1 and P2 are expressed constitutively which is in agreement with the high levels of polymerase I observed previously [1]. When pC J63 is introduced into the UV-resistant strain the con- stitutive expression only occurs from the P2 pro- motor whereas in the same strain carrying pC J93, expression is under the control of both the pro- motors. Hence maximum transcription of the polA-galK fusion is observed.

I

02

~alK J J l l l l l l / / l ~ l l l l l l i / l l l l l l l l l l l l / l

j ~alK 7 1 1 1 1 / i i I l l / I l l r l l l l l l l l l / l / l 1 1 1 I I

polA

llJJp / fl I~ f i lJf4f j if jf jf fjf j lJlJJiJ

Data presented (Table 1) demonstrate that the UV-sensitive mutation (strain SA322) has no effect on the activity of polA promotors expressed either on pC J63 or pC J93. The results suggest that the second mutational site in SA236 has no significant role in the regulation of the polA gene.

It would be interesting to study the influence of the mutant gene the expression of poiA-galK fu- sion with a derivative of pC J93 having its dnaA binding site affected by local mutagenesis without changes in the polA promotors themselves.

Acknowledgement

We wish to thank Dr. C.M. Joyce for providing us with the plasmids and one of the authors (S.I.A.) wishes to thank the Commission of the European Communities for providing a Training Fellowship during which the work presented in this paper was carried out. The authors also wish to thank Dr. B.J. Bachmann for providing E. coli strains.

References

1 Ahmad, S.I., A. Atkinson and A. Eisenstark (1980) Isola- tion and characterization of a mutant of Escherichia coil

K12 synthesising DNA polymerase I and endonuclease I constitutively, J. Gen. Microbiol., 117, 419-422.

2 Bachmann, B.J., and K. Brooks Low (1980) Linkage map of Escherichia coil K12, Edn. 6, Microbiol. Rev., 44, 1-56.

3 Fuller, R.S., B.E. Funnell and A. Kornberg (1984) The dnaA protein complex with the Escherichia coli chromosomal replication origin (oriC) and other DNA sites, Cell, 38, 889-900.

4 Glickman, B. (!974) The role of DNA polymerase I in pyrimidine dimer excision and repair replication in Escherichia coli KI2 following ultraviolet irradiation, Biochim. Biophys. Acta, 335, 115-122.

5 Joyce, C.M., S. Kelley and D.F. Grindley (1982)Nucleotide

sequence of the Escherichia coli polA gene and primary structure of DNA polymerase I, J. Biol. Chem., 257, 1958-1964.

6 Kornberg, A. (1974) DNA Synthesis, Freeman, San Francisco.

7 Maniatis, T., E.F. Fritsch and F. Sambrook (1982) Molecular Cloning, Cold Spring Harbor, 250 pp.

8 McKenney, K., H. Shimatake, D. Court, U. Schmeisser, C. Brady and M. Rosenberg (1981) in: J.G. Chirikijian and T.S. Papas (Eds.), Gene Amplification and Analysis, Vol.

81

2, Elsevier, Amsterdam, pp. 383-415. 9 Miller, J.H. (1972) Experiments in Molecular Genetics,

Cold Spring Harbor Laboratory, New York. l00 'Cal laghan, C.H., A. Morris, S. Kirby and A.P. Shingler

(1972) Antimicrob. Agents Chemother., l, 283-288. 11 Ward, D.F., and N.E. Murray (1980) Construction and

characterization of Escherichia coli K 12 polA-lacZ gene fu- sion, J. Bacteriol., 142, 962-972.

Communicated by F.H. Sobels