Mutational DNA Base Sequence Changes in Plasmid. Damaged at a Specific Region by Ultraviolet Light

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1 Mutational DNA Base Sequence Changes in Plasmid Damaged at a Specific Region by Ultraviolet Light KOICHI TAKIMOTO Radioisotopes Laboratory, Faculty of Agriculture, Yamaguchi University, Yoshida, Yamaguchi 753, Japan (Received December 18, 1985) (Revised version, accepted April 12, 1986) Plasmid/UV-mutagenesis/Base change/transition/pyr(6-4)pyo photoproduct Mutational nucleotide sequence changes by ultraviolet light (UV) in the gene for Escherichia coli CAMP receptor protein (CRP) were analyzed. Transitions were most the frequent mutations and occurred at pyrimi dine-pyrimidine sequences. Most transitions were GC to AT base changes at 3' cytosine of 5'-pyrimidine cytosine-3' sequences, supporting the concept that pyrimidine(6-4)pyrimidone, Pyr(6-4)Pyo, photoproduct is a principal pre-mutagenic lesion for transition mutations induced by UV. Transversion and frameshift mutations were also found. INTRODUCTION Ultraviolet light is an effective mutagen for E. coli. UV-induced mutation is presumably due to the expression of SOS responses, requiring a set of genes, notably reca, lexa and umucd1-4). Investigation of the changes in nucleotide sequence in mutation leads to an under standing of hotspots for mutation and the spectrum of UV-mutagenesis. Genetic analysis based on the production or reversion of nonsense mutations has been done at limited sites in the genes). Direct assays of nucleotide sequence over the target gene for the detection of any type of mutations have been reported in some limited genes, such as lac promoter, cl, lacl and tetracycline gene S6-9). Recently, it has been suggested that a UV photoproduct other than cyclobutyl pyrimidine dimer is the main pre-mutagenic DNA lesion responsible for most UV induced mutagenesis7,10,11) Further investigation of nucleotide sequence changes in UV induced mutation in a variety of genes are needed to understand hotspots for mutation, pre mutagenic lesions and the general features of UV-mutagenesis. Site directed mutagenesis is based on the introduction of a mutation to a specific region on the gene9) which is available for the analysis of mutation. I have investigated mutational nucleotide sequence changes in the crp gene for analysis of the specificity of UV-mutagenesis using the direct mutagenesis method. The assay system used is suitable for direct analysis of nucleotide sequence changes.

2 MATERIALS AND METHODS Plasmid and bacteria Plasmid pha7, comprising 5.2k base pairs (bp), carries E. coli camp receptor protein gene (crp) and ampicillin resistance gene. The coding region of the crp gene consists of 627 by and is located between EcoRI and Hindlll sites on pha7. The crp gene fragment has two cleavage sites for HapII. E. coli pp4712), which is Crp and confers Lac phenotype, was used throughout the experiment. Materials Restriction endonucleases, T4 DNA ligase and T4 polynucleotide kinase were obtained from Takara Shuzo Co. [y-32 P] ATP ( Ci/mmol) was purchased from Amersham. Mutation induction The pha7, irradiated with lk J/m2 of UV under germicidal lamps (principally 254 nm) and unirradiated pha7, were digested with EcoRI and HindIll. The irradiated crp gene fragment (the target fragment) and unirradiated large fragment were ligated to construct the plasmid with UV lesions on the target region (Fig. 1). UV-irradiated cells were incubated at 37 C for 20 min in the dark for the induction of SOS responses. The E. coli cells transformed with the plasmid were grown on lactose-macconkey agar plates containing ampli cillin (50 pg/ml). The E. coli cells transformed with an intact pha7 to Lac+ phenotype form red colonies, while the plasmid carrying the defective crp gene by mutation does not transform to Lac+ and white colonies appear because the lactose operon is inactive. Base sequence analysis The crp target fragment was digested with HapII. Each fragment isolated was labeled with [,y_12p] ATP using T4 polynucleotide kinase. The labeled DNA was separated into single-stranded DNA by polyacrylamide gel electrophoresis for strand separa tion 13). DNA sequence was determined by the Maxam-Gilbert chemical reaction method, and by 6% and 20% thin polyacrylamide gel electrophoresis13) Fig. 1. Reconstruction of plasmid DNA specifically damaged at the target segment. AP r and crp indicate ampicillin resistance gene and the coding region for CRP, respectively.

3 Fig. 2. Enhanced transformation efficiency in UV-irradiated E. coli pp47 cells. UV-irradiated cells were incubated at 37C for 20 min in the dark and then transformed with UV-irradiated (1k J/m2) or unirradiated plasmid. Transformation efficiency is the normalized ratio of transformants given by the irradiated plasmid in irra diated cells to that in unirradiated cells. Fig. 3. Nucleotide sequence changes in the coding region for CRP. The numbers indicate the bases from the first base of initiation of mrna synthesis.

4 RESULTS The E. coli cells irradiated with different fluences of UV were transformed with UV irradiated (lk J/m2) or unirradiated pha7. About a three-fold increase in transformation efficiency was observed in the cells irradiated with 25 J/m2 of UV (Fig. 2), indicating an induc tion of SOS repair system. When reconstructed plasmid was used, the transformation efficiency was about two times higher in irradiated (25 J/m2) cells than in unirradiated cells. Background mutation frequency (Lac and ampicillin resistant phenotype) was less than The fre quency of mutants produced by transformation with the reconstructed plasmid was about 8 x 10-3 in unirradiated cells and 1.8 x 10-2 in irradiated (25 J/m2) Cells. Forty-four mutant colonies were isolated. Plasmids isolated from mutants were digested with restriction endonucleases. Large deletions and insertions at the target segment and other regions, and a loss of restriction sites were found in thirty-two plasmids after gel electro phoresis. The reason for these large structural changes is not known. These plasmids were excluded from further analysis because there was no way to distinguish between genuine UV induced mutations and artifacts produced in the process of manipulation of DNA. Fourteen mutation sites were found in the coding region for CRP after twelve mutant plasmid DNAs were sequenced. Single-base changes occurred at eleven sites and frameshift type changes at three sites (Fig. 3). Nine mutations among single-base change mutations were tran sition type and two were transversion type. All transition mutations occurred at Pyr-Pyr sequences. Six transition mutations were GC to AT base changes and three were AT to GC. All the GC to AT transitions appeared at 3'C of 5'-Pyr-C-3' sequences. Three AT to GC base changes was found at thymine-thymine base pairs. In the frameshift type mutations, two were one base deletions and one was a single base insertion. DISCUSSION All the transition mutations detected appeared at Pyr-Pyr sequence. There is a strong correlation between transition mutations and Pyr-Pyr sequences, as reported earlier"). Of nine transition mutations, six were GC to AT base changes. The GC to AT transitions were also predominant in UV-mutagenesis of lacl gene") Base changes in transition mutations occurred at 3'pyrimidine of adjacent pyrimidines except one transition, consistent with a previous report 14). Brash and Haseltine1o) suggested that the pyrimidine dimers are not a main pre-mutagenic lesions caused by UV and that Pyr(6-4)Pyo photoproducts, minor photoproducts, are mutagenic. The photoproducts occur most frequently at TC, at some CC and infrequently at TT sequences"). Wood et al.7,14) have also strongly suggested that the photoproduct is a main pre-mutagenic UV lesion responsible for most transition mutations. Also in the result obtained here, of nine transition mutations, six occurred at 3'C of 5'-T(C)-C-3' sequences, indicating that a principal DNA lesion for transition mutation induced by UV is presumably Pyr(6-4)Pyo photoproduct. AT to GC base changes detected at three sites occurred at the sequence of two or more adjacent thymines. Since the formation of Pyr(6-4)Pyo photoproduct at TT sequence is infrequent, the base change muta

5 tions were presumably due to thymine dimers, suggesting that thymine dimers are not ruled out as pre-mutagenic DNA lesions although they are not the principal lesion leading to base change mutation. Wood 14) has detected AT to GC transitions at thymine-thymine sequences. The frequency of transversion mutation was low, as previously reported? 8). Miller8) has reported that frameshift type mutations were found at the sequence where two or more AT base pairs were repeated. In the present study, one frameshift mutation was detected at repeated AT base pairs. Wood and Hutchinson") have demonstrated that the frameshift muta tions significantly occurred in unirradiated phage lambda grown in UV-irradiated E. coli cells. I was unable to determine whether frameshift mutations detected here were due to targeted or non-targeted mutagenesis. ACKNOWLEDGEMENTS The author wishes to thank Dr. H. Aiba, Tsukuba University, for kindly donating plasmid and for valuable suggestions, and Dr. K. Ishizaki, Kyoto University, for valuable discussions and criticism throughout the work. A part of this work was carried out at The Radiation Biology Center, Kyoto University. This work was supported by Grant-in-Aid from Ministry of Educa tion, Science and Culture, Japan and by Mitsuhisa Memorial Cancer Research Fund. REFERENCES 1. Witkin, E. M. (1976) Ultraviolet mutagenesis and inducible DNA repair in Escherichia coll. Bacteriol. Rev. 40: Hall, J. D. and Mount, D. W. (1981) Mechanisms of DNA replication and mutagenesis in ultraviolet irradiated bacteria and mammalian cells. Prog. Nucl. Acid Res. Mol. Biol. 25: Little, J. W. and Mount, D. W. (1982) The SOS regulatory system of Escherichia coli. Cell 29: Walker, G.C. (1984) Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli. Microbiol. Rev. 48: Miller, J. H. (1982) Carcinogens induce targeted mutations in Escherichia coll. Cell 31: LeClerc, J. E. and Istock, N. L. (1982) Specificity of UV mutagenesis in the lac promoter of M13lac hybrid phage DNA. Nature 297: Wood, R. D., Skopek, T. R. and Hutchinson, F. (1984) Changes in DNA base sequence induced by targeted mutagenesis of lambda phage by ultraviolet light. J. Mol. Biol. 173: Miller, J. H. (1985) Mutagenic specificity of ultraviolet light. J. Mol. Biol. 182: Livneh, Z. (1983) Directed mutagenesis method for analysis of mutagen specificity: Application to ultraviolet-induced mutagenesis. Proc. Natl. Acad. Sci. USA 80: Brash, D. E. and Haseltine, W. A. (1982) UV-induced mutation hotspots occur at DNA damage hot spots. Nature 298: Haseltine, W. A. (1983) Ultraviolet light repair and mutagenesis revised. Cell 33: Aiba, H., Fujimoto, S. and Ozaki, N. (1982) Molecular cloning and nucleotide sequencing of the gene for E. coli camp receptor protein. Nucl. Acids Res. i0: Maxam, A. M. and Gilbert, W. (1980) In "Methods in Enzymology", Vol. 65, Ed. L. Grossman and K. Moldave, pp , Academic Press, New York. 14. Wood, R. D. (1985) Pyrimidine dimers are not the principal pre-mutagenic lesions induced in lambda phage DNA by ultraviolet light. J. Mol. Biol. 148: Wood, R. D. and Hutchinson, F. (1984) Non-targeted mutagenesis of unirradiated lambda phage in Escherchia coli host cells irradiated with ultraviolet light. J. Mol. Biol. 173: