Proc. Natl. Acad. Sci. USA Vol. 83, pp. 6945-6949, September 1986 Genetics The C-C (6-4) UV photoproduct is mutagenic in Escherichia coli (UV mutagenesis/pyrimidine dimer/5-methylcytosine/targeted mutagenesis/mutational specificity) BARRY W. GLICKMAN*t, ROEL M. SCHAAPERt, WILLIAM A. HASELTINEt, RONNIE L. DUNNt, AND DOUGLAS E. BRASH§¶ *Biology Department, York University, 4700 Keele Street, Toronto, Canada M3J 1P3; tLaboratory of Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709; SLaboratory of Biochemical Pharmacology, Dana-Farber Cancer Institute, Boston, MA 02115; and §Laboratory of Human Carcinogenesis, Building 37, National Cancer Institute, Bethesda, MD 20892 Communicated by Richard B. Setlow, June 2, 1986 ABSTRACT Mutation induced by ultraviolet light is pre- explanation for the photoreactivation of UV-induced dominantly targeted by UV photoproducts. Two primary mutagenesis that avoids an obligatory role for cyclobutane candidates for the premutagenic lesion are the cyclobutane dimers in directly targeting mutation. pyrimidine dimer and the less frequent (by a factor of 10) To determine whether the (6-4) photoproduct is capable of pyrimidine-pyrimidone (6-4) photoproduct. Methylation of the targeting mutation, we specifically increased the yield of (6-4) 3'-cytosine in the sequence 5' CCAGG 3' reduces the yield of photoproducts at particular C-C sequences. To achieve this, (6-4) lesions, but not of cyclobutane dimers, at these sites. By we took advantage of the observation of Brash and Haseltine taking advantage of mutants deficient in cytosine methylation, (3) that the (6-4) photoproducts form less efficiently at sites we show here that at the three sites in the lacI gene of of cytosine methylation. At the sequence 5' CCAGG 3', the Escherichia coli having this sequence, the specific increase in second cytosine is normally methylated, but in Dcm- strains the formation of the (6-4) photoproducts is accompanied by a this cytosine is unmethylated (24). We measured the frequen- concomitant increase in mutation. At each site, a GC to APT cy of UV-induced nonsense mutations and the formation of transition results in an amber mutation. In the unmethylated UV-induced photoproducts at the three such sites in the E. state, these sites become among the most frequent nonsense coli lacI gene, as well as at nearby sites not subject to mutations recovered. We conclude that the (6-4) photoproduct cytosine methylation. In the absence of methylation, the constitutes a major premutagenic lesion in E. coli. yield of C-C (6-4) photoproducts was increased 2.4-fold, whereas the yield of C-C cyclobutane pyrimidine dimers was Mutagenesis by ultraviolet light appears to be targeted by slightly decreased. The yield of nonsense mutations in- specific DNA lesions (1-10). The sites of mutation indicate creased 3-fold. The concomitant increase in (6-4) photoprod- that dipyrimidines constitute the major mutational target. ucts and mutational yield at these sites suggests the (6-4) Two major dipyrimidine photoproducts have been identified photoproduct to be a premutational lesion. in DNA. These are the cyclobutane pyrimidine dimer (11) and the pyrimidine-pyrimidone (6-4) photoproduct [6-4'-(pyrimi- MATERIALS AND METHODS din-2'-one)-5-aminocytosine] (12-15). It has been generally believed that the cyclobutane dimers are the principal Strains, Media, and the lacI System. Unless otherwise premutagenic lesion. This belief was based on the observa- stated, the materials and techniques are as described by Todd tion of increased mutation in strains defective in repair of and Glickman (4). Strain NR3989 is, with the exception ofthe cyclobutane dimers and was fostered by the loss of mutation dcm mutation, the isogenic partner of NR3835 [F' pro- following photoreactivation treatment, a process specific to lac/A(pro-lac), ara-, thi, trpE9777]. The Dcm- derivative the reversal of these lesions (16, 17). More recently, the was constructed by introducing the dcm mutation (24) via potential of cyclobutane dimers to target mutation in Esch- P1-mediated transduction into a his- derivative of NR3835 erichia coli was demonstrated by the single-stranded transfer and selecting for His+ transductants. These transductants of irradiated DNA into a recipient cell (6, 18) and in the were screened and the dcm character was monitored by single-stranded bacteriophage S13 (7). EcoRII and BstNI (Bethesda Research Laboratories) restric- Evidence has emerged, however, that challenges the ex- tion analysis of the plasmid pMC1 grown in this strain. BstNI clusiveness of cyclobutane dimers in targeting mutation in cuts the sequence 5' CCAGG 3' regardless of methylation, double-stranded DNA. There appears, for example, to be a whereas EcoRII cuts at this sequence only when' it is better correlation between the distribution of (6-4) photo- unmethylated. In addition, the presence of the dcm mutation products in the DNA and the position of mutation than is the was confirmed by the resulting absence of the mutational hot case for cyclobutane dimers (3). In addition, the specific spots associated with deamination at methylated 5' CCAGG induction of thymine-containing cyclobutane dimers by 3' sequences (25). UV treatment, mutant selection, and acetophenone plus UV light (313 nm) does not increase the mutant analysis were carried out as described by Coulondre yield of transition mutations (5, 19), the major class of and Miller (20) as modified by Todd and Glickman (4). mutation induced by UV light (10, 20), nor did photoreactiva- UV Photoproducts at Single Base Pairs. To measure tion of cyclobutane dimers from phage X DNA reduce cyclobutane pyrimidine dimers and pyrimidine-pyrimidone mutagenesis (21). Photoreactivation reverses mutagenesis at (6-4) photoproducts at site AmlS, plasmid pMC1 (26) purified the his locus of E. coli less effectively than it reverses the from wild-type and Dcm- E. coli was 32P-labeled at the 5' lethal lesion (22). Finally, the observation that photoreactiva- termini of the unique Mlu I site (located at base pair 380 in the tion prevents the induction of the umuC, -D gene product lacI gene) by the forward kinase reaction (27). The singly required for UV mutagenesis (23) provides an alternative end-labeled 180 base-pair Mlu I to BstEII fragment was then isolated by the methods of Maxam and Gilbert (28). For site The publication costs of this article were defrayed in part by page charge Am6, the 430-base-pair Mlu I to HincII fragment was first payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. STo whom reprint requests should be addressed. 6945 Downloaded by guest on September 24, 2021 6946 Genetics: Glickman et al. Proc. Natl. Acad. Sci. USA 83 (1986) Table 1. Mutation frequencies following a UV dose of 125 j/m2 Table 2. Influence of cytosine methylation on mutation induction Frequency of NR3835 (Dcm+) NR3989 (Dcm-) at methylatable and nonmethylatable sites (1.1 ± 0.3) x 10-4 (1.0 ± 0.2) x 10-4 No. of x2 Significance, acI-,otad Site Strain isolates* value P Dcm-/Dcm't lacIamber (10.0 ± 4.5) x 10-6 (15.0 ± 5.3) x 10-6 lacIochre ( 6;0 ± 2.7) x 10-6 (10.0 ± 3.9) x 10-6 AM5 wt 53 0.2 NS 1.1 The survival in both strains was 2.7%. These results are the Dcm- 25 average of 10 and 6 cultures in strains NR3835 and NR3839, AM6 wt 33 38 <0.001 3.4 respectively. The range in values is the 95% confidence interval. The Dcm- 49 average spontaneous mutation frequency is 2.4 x 10-6 for both the AM15 wt 26 20 <0.001 2.5 Dcm+ and Dcm- strain. Dcm- 28 AM16 wt 75 0.6 NS 0.83 isolated. This was then cut with Hpa II; the 5' termini labeled Dcm- 27 and, finally, the 250-base-pair Hpa II to Taq I fragment singly AM33 wt 70 0.08 NS 1.0 end-labeled at the Hpa II site (located at base pair 67 in the Dcm- 31 lacIgene) was isolated. Plasmid pMC1 contains slow sites for AM34 wt 16 7.0 <0.01 2.3 Nar I digestion. Hence, in the case of Am34, the DNA had Dcm- 16 to be digested extensively with this enzyme (New England Methylatable sites are italicized. NS, not significant (P > 0.5); wt, Biolabs). The 3' termini were labeled with the Klenow wild type. *Number of independent isolates from a collection of 800 and 349 fragment ofPol I, and a 4-kilobase doublet was isolated from UV-induced amber mutations in the Dcm' and Dcm- strains, SeaPlaque agarose (Marine Colloids, Rockland, ME). After respectively. cutting with Mlu I, the 640-base-pair Nar I to Mlu I fragment tCalculated as the ratio of fraction of mutants occurring at that site singly end-labeled at the Nar I site (located at base pair 1019 in the Dcm- strain and the Dcm+ strain. in the lacl gene) was then isolated. End-labeled DNA was irradiated with 254-nm light (500-5000 J/m2) and the frequency ofcyclobutane pyrimidine dimer-specific glycosylase plus apurinic endonuclease (ref. dimers and pyrimidine-pyrimidone (6-4) photoproducts at 30; gift ofJ. Lippke) or T4 endonuclease V (ref. 31; gift of A. single base pairs was measured as described (29). Briefly, Ganesan). The (6-4) photoproducts were detected by incu- cyclobutane dimers were detected by incubating irradiated bating irradiated DNA with 1 M piperidine for 20 min at 900C. DNA with an excess of Micrococcus luteus pyrimidine This procedure cleaves the phosphodiester bond of all (6-4) 25 r 20 W a)0 a) 15 * cJ 0 U)0 c 0' 1 0 0~ * 5 0 Amber sites FIG. 1.