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 / 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 induced by light is pre- explanation for the photoreactivation of UV-induced dominantly targeted by UV photoproducts. Two primary mutagenesis that avoids an obligatory role for 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'- 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 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 -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

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a) 15 * cJ

0 U)0

c 0' 1 0 0~ *

5

0

Amber sites FIG. 1. A comparison of the distribution of lacI amber mutants induced by UV (125 J/m2) in a Dcm+ and Dcm- strain of E. coli. Open and solid bars give the spectrum of 800 and 349 amber mutants recovered in the Dcm+ and Dcm- strains, respectively. *Bracketed comparisons indicate statistically significant differences (see Table 2). Downloaded by guest on September 24, 2021 Genetics: Glickman et al. Proc. Natl. Acad. Sci. USA 83 (1986) 6947 1 2 3 4 5 6 7 8 9 10 11

cc _I -CC

CT- _ TT-M -N _ i i CC- s-1-1 -CC

,0- for-*, * -- a a" Am ' CC*/zc_ Am - cc Am15

C

TC - *o- - TC Cc-- "- -C Cg3 -S: 5'CTIMGCAIGGATGAn3*AGGATG'AIIGGTGGAAG CTGgGCAjJAATGTT: 3' 1 2 3 4 5 6 7 8 9 10 11 CC- 1 W _ ai *0 00_0 1-CCc wF CC -

CTTW- asrn _,cc CT CT

TC7CT CT F ^ - TC TC Am33 TC a"iias - TC Am33 Am34 CC*/CC -CC' CC Am34

TC- A

mA410. avj TC

5' ACCAGCGTGGACCGCTTGCTGCAACTCTCTC AGGGCC* AGGCGGTGAAGGGCAATCAGCT 3'

FIG. 2. Influence of cytosine methylation on the distribution and frequency of cyclobutane pyrimidine dimers and pyrimidine-pyrimidone (6-4) photoproducts in the lad gene. (Upper) Site Am15. (Lower) Site Am34. Lanes: 1 and 2, incubation with cyclobutane dimer-specific endonuclease after 500 J/m2; Dcm' and Dcm-, respectively; 3, guanine + adenine Maxam-Gilbert sequencing reaction; 4 and 5, cytosine + thymine sequencing reaction; Dcm+ and Dcm-, respectively; 6-11, incubation with hot piperidine to reveal (6-4) lesions; 6 and 7, 500 J/m2 254-nm light; Dcm+ and Dcm-, respectively; 8 and 9, 2000 J/m2; Dcm+ and Dcm-, respectively; 10 and 11, 5000 J/m2; Dcm+ and Dcm-, respectively.

photoproducts formed in dinucleotides, including the C-T RESULTS (6-4) photoproduct not formed in B-DNA (32). Maxam- and Gilbert sequencing reactions were performed as markers. Survival and Mutagenesis. Treatment of the Dcm' Samples were analyzed as described and individual bands Dcm- strains of E. coli with a UV dose at 125 J/m2 resulted were quantitated by Cerenkov counting after being cut out of in similar levels of lethality and mutagenesis (Table 1). The the gel and/or by densitometric scanning of the autoradi- overall induction of lacI mutations was -44-fold above the ograms in comparison to known standards. The percentage of spontaneous frequency, while the induction of nonsense initial molecules carrying scissions at a particular site were mutations was -1000-fold. Hence, few, if any, of the mutants computed (3, 33). taken for the analysis of the mutational spectrum would be Downloaded by guest on September 24, 2021 6948 Genetics: Glickman et al. Proc. Natl. Acad. Sci. USA 83 (1986) Table 3. Influence of cytosine methylation on the distribution of The specific effect of methylation on C-C (6-4) photoprod- UV-induced damage at specific sites in the lacI gene uct frequency was also seen at the two other methylation sites Cyclobutane dimers (6-4) photoproducts in the lacI gene, Am6 and Am34. Site Am34 is shown in Fig. 2 (Lower). The quantitation of the cyclobutane dimers and Dcm-/ Dcm-/ the (6-4) photoproducts in both the Dcm' and Dcm- strains Site Dcm' Dcm- Dcm' Dcm' Dcm- Dcm' are summarized in Table 3. In general, the state of cytosine AM5 0.47 0.44 0.9 0.21 0.27 1.3 methylation had little or no effect on the number of AM6 0.22 0.28 1.3 0.04 0.10 2.5 cyclobutane pyrimidine dimers produced. In contrast, (6-4) AMJS 0.50 0.23 0.5 0.05 0.14 2.8 photoproducts increased significantly (range 2.0- to 2.8-fold) AM16 1.3 1.2 0.9 0.96 1.1 1.2 in the absence of methylation. Nearby sites, not subject to AM33 1.1 0.84 0.8 0.86 0.59 0.7 dcm methylation, but capable of producing an amber muta- AM34 0.1 0.07 0.7 0.04 0.08 2.0 tion by a G-C -+ APT transition, showed no increase in the amount of (6-4) lesion (Table 3), nor was mutagenesis Methylatable sites are italicized. Numbers are percentages of molecules carrying a cyclobutane dimer or a (6-4) photoproduct at enhanced at these sites (Table 2). specific sites following a dose of 500 J/m2. Repeated measurements displayed a variance of <20%. DISCUSSION Three sites in the lacI gene can be converted to an amber spontaneous in origin. This includes mutation at sites Am6, mutation by a G-C -. APT transition at positions normally Aml5, and Am34, which, because of the spontaneous deam- containing 5-methylcytosine (5' CCAGG 3' -*5' CTAGG 3'). ination of the 5-methylcytosine residues, are hot spot sites in Since the yield of (6-4) photoproducts was previously report- the spontaneous nonsense spectrum (25). The UV treatment ed to be low at a C-C sequence where the 3' cytosine is results in a >100-fold increase over the spontaneous levels of methylated (3), these sites provide an opportunity to distin- mutation at these sites. guish between the mutagenic effects of cyclobutane dimers In this study, 800 and 347 amber mutations recovered in the and (6-4) photoproducts. We therefore examined the muta- wild-type and Dcm- strains, respectively, were character- tional spectra produced by UV light in an isogenic pair of ized (Fig. 1). The mutational spectrum observed in the Dcm' strains differing only in their ability to methylate the cytosine strain is similar to- that reported earlier for this strain (4, 34) at the target sequence and compared the induction of muta- and to that reported by Miller (10). Most mutations occur at tion with the production of cyclobutane dimers and (6-4) sites of potential dipyrimidine lesions and are recovered as photoproducts at the same sites. G-C -* A-T transitions. Similar results were found in the The results show that mutation at normally methylated analysis of mutations induced in the Dcm- strain. However, sites is significantly enhanced in the absence of methylation relative mutation frequencies at the normally methylated (Fig. 1; Table 2). The increase in the production of the (6-4) sites Am6, Aml5, and Am34 were increased in the Dcm- photoproduct (Fig. 2; Table 3) at these three sites is accom- strain at least 3-fold (Table 2); in each case, this increase is panied by a parallel increase in mutation. In contrast, the statistically significant. yield of cyclobutane dimers at these sites is unaltered or Quantitation of DNA Photoproducts. Fig. 2 (Upper) shows slightly diminished. The correlation between the increase in the distribution of cyclobutane pyrimidine dimers and py- mutation at these sites and the increase in the (6-4) rimidine-pyrimidone (6-4) photoproducts in a region of the photoproduct leads us to the conclusion that the (6-4) lacl gene extending from base pair 403 to base pair 459 and photoproduct targets the G-C -* APT transitions observed at including the site Aml5. The locations ofcyclobutane dimers these sites; This conclusion is not dependent on the action of are indicated by bands in the lanes containing samples treated excision repair, since similar spectra were obtained in a with dimer-specific endonuclease (lanes 1 and 2). The inten- UvrB- strain (data not shown). sity of a band reflects the frequency of scission at the We recognize that other interpretations of these data are corresponding base and, hence, the frequency of dimers at possible. For example, we are unable to exclude the possi- that site in the population of irradiated DNA molecules. As bility that an as yet uncharacterized dipyrimidine lesion shown in Fig. 2 Upper, the intensities of dimer bands in the behaves in a manner similar to that observed for the (6-4) Dcm+ strain (lane 1) are equal to those in the Dcm- strain photoproduct at sites of 5-methylcytosines. We are equally (lane 2). These bands represent sites of T-T, T-C, C-T, and unable to exclude the possibility that 5-methylcytosine- C-C cyclobutane pyrimidine dimers and include site Ami5 at containing cyclobutane dimers fortuitously instruct DNA base pair 419. The methylation status of the Aml5 site is polymerase to correctly insert a guanine opposite the 5- confirmed in the sequencing lanes. Cytosine methylation methylcytosine rather than incorrectly insert an adenine inhibits the cytosine plus thymine Maxam-Gilbert sequenc- residue. We are, however, unaware of any experimental ing reaction (28), and at base pair 419 the cytosine plus observations that would lend support to the above alternative band in the Dcm- strain (lane 5) explanations and therefore prefer the interpretation that the thymine sequencing appears correlation between the enhanced production of (6-4) but is absent in the wild-type strain (lane 4). photoproducts and mutation reflects the role of these lesions As revealed by piperidine hydrolysis, the distribution of in targeting the G-C -* A-T transition. T-C and C-C (6-4) photoproducts in this DNA fragment is The structure of the (6-4) photoproduct may account for identical in Dcm+ and Dcm- strains with the exception of a the observed G-C -k APT specificity. An examination of single site (lanes 6 and 7). At base pair 419, the site of models of DNA containing the C-C (6-4) photoproduct (15) mutation AmiS, the C-C (6-4) lesion band is 3-fold more suggests that the 5'-pyrimidine ring of the lesion can still pair intense in Dcm- than in Dcm+. (In 5'- but not 3'-labeled with guanine. The 3'-pyrimidone ring, however, can no DNA, the Dcm+ cleavage product also migrates -1 base longer base pair with guanine because its amino group has more slowly than the Dcm- product, presumably because of been transferred to the 5'-pyrimidine ring, and the angle of the additional methyl group.) To magnify the very low yield the plane of the ring will not permit base pairing in the B-form of C-C (6-4) lesions at the methylated Aml5 site, we also of DNA. The local distortion at the 3'-pyrimidone ring is measured (6-4) lesions at higher doses (lanes 8-11). We found similar to that seen at an apyrimidinic site. It has been shown that the C-C (6-4) lesion is indeed produced at methylated previously that E. coli DNA polymerase I preferentially sites, but at a lower frequency. inserts adenine residues at apyrimidinic sites (35). This Downloaded by guest on September 24, 2021 Genetics: Glickman et al. Proc. Natl. Acad. Sci. USA 83 (1986) 6949

specificity is also observed for apurinic sites in vivo (36, 37). 3. Brash, D. E. & Haseltine, W. A. (1982) Nature (London) 298, The insertion of an adenine opposite the 3'-pyrimidone ring 189-192. of the C-C (6-4) lesion would explain the predominance of 4. Todd, P. A. & Glickman, B. W. (1982) Proc. Natl. Acad. Sci. G-C -- APT transitions we observe at sites of the formation of USA 79, 4123-4127. this 5. Fix, D. F. & Bockrath, R. C. (1983) Proc. Natl. Acad. Sci. photoproduct. This mechanism is consistent with the USA 80, 4446-4449. known requirement for the induction of SOS repair for UV 6. Kunz, B. A. & Glickman, B. W. (1984) Genetics 106, 347-364. mutagenesis (17, 38), since mutation at depurinated sites is 7. Tessman, I. (1985) Proc. Natl. Acad. Sci. USA 82, 6614-6618. SOS dependent (39). 8. Wood, R. D. & Hutchinson, F. J. (1984) J. Mol. Biol. 173, Our results indicate that the (6-4) lesion plays a major role 293-305. in UV-induced G-C -* APT transitions in the lad gene. 9. Bridges, B. A. & Woodgate, R. (1984) Mol. Gen. Genet. 1%, Mutations that arise at the three amber sites only in the 364-366. unmethylated state are likely to be targeted by (6-4) lesions. 10. Miller, J. H. (1985) J. Mol. Biol. 182, 45-68. Because this increase is -3-fold, it follows that -"67% of the 11. Beukers, R. & Berends, W. (1960) Biochim. Biophys. Acta 41, mutations at these sites must be due to 550-557. these lesions. In 12. Varghese, A. J. & Wang, S. Y. (1967) Science 156, 955-957. addition, as shown in both Fig. 2 and Table 3, some (6-4) 13. Varghese, A. J. & Patrick, M. H. (1969) Nature (London) 223, photoproducts are produced even in the Dcm' strain. Wheth- 299-300. er these lesions reflect unmethylated sites or the reduced 14. Lippke, J. A., Gordon, L. K., Brash, D. E. & Haseltine, formation of the (6-4) lesion in the presence of methylation W. A. (1981) Proc. Natl. Acad. Sci. USA 78, 3388-3392. cannot be determined. Nonetheless, the formation ofthe (6-4) 15. Franklin, W. A., Doetsch, P. W. & Haseltine, W. A. (1985) lesion at these sites in the Dcm' strain makes it possible that Nucleic Acids Res. 13, 5317-5325. a significant fraction of the remaining mutations are also 16. Witkin, E. M. (1966) Science 152, 1345-1353. targeted by (6-4) lesions. We therefore conclude that more 17. Witkin, E. M. (1976) Bacteriol. Rev. 40, 869-907. than 70% of the at 18. Lawrence, C. W., Christensen, R. B. & Christensen, J. R. mutations these three sites are targeted by (1985) J. Bacteriol. 161, 767-768. (6-4) lesions. Any additional contribution of cyclobutane 19. Wood, R. D., Skopek, T. R. & Hutchinson, F. J. (1984) J. dimers to the targeting of these mutations need not be Mol. Biol. 173, 243-291. extensive. 20. Coulondre, C. & Miller, J. H. (1977) J. Mol. Biol. 117, With respect to other potential (6-4) target sites in the lad 243-291. gene, there are only 12 sites where a G&C -* A-T transition at 21. Wood, R. D. (1985) J. Mol. Biol. 184, 577-585. a potential pyrimidine-pyrimidine target can produce an 22. Yamamoto, K., Shinagawa, H. & Ohnishi, T. (1985) Mutat. amber mutation. Yet mutations observed at the three sites Res. 146, 33-42. studied here (25% of the available sites) accounted for 26% of 23. Brash, D. E. & Haseltine, W. A. (1985) J. Bacteriol. 163, all the amber in 460-463. mutations induced the Dcm- strain. Despite 24. Marinus, M. G. (1973) Mol. Gen. Genet. 127, 47-55. considerable site-to-site variation in mutation frequencies in 25. Coulondre, C., Miller, J. H., Farabaugh, P. J. & Gilbert, W. the lad gene, the Am6, Am15, and Am34 sites are not (1978) Nature (London) 274, 775-780. atypical. All three, when in the unmethylated state, fall 26. Calos, M. P., Johnsrud, L. & Miller, J. H. (1978) Cell 13, within the range observed at other potential G-C -- AT target 411-418. sites. It is thus probable that a similar contribution to 27. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1980) Molecular mutagenesis is made by the (6-4) lesions we observe at other Cloning: A Laboratory Manual (Cold Spring Harbor Labora- potential target sites. The contribution may be even greater tory, Cold Spring Harbor, New York). at sites of T-C (6-4) photoproducts since these lesions are 28. Maxam, A. M. & Gilbert, W. (1980) Methods Enzymol. 65, induced in abundance than 499-560. greater the C-C (6-4) 29. Brash, D. E., Franklin, W. A., Sancar, G. B., Sancar, A. & photoproducts (3) and should also generate G-C -k APT Haseltine, W. A. (1985) J. Biol. Chem. 260, 11438-11441. transitions. The major fraction of G-C -* NT base substitu- 30. Haseltine, W. A., Gordon, L. K., Lindan, C. P., Grafstrom, tions occurring in the lad forward mutation assay may R. H., Shaper, N. L. & Grossman, L. (1980) Nature (London) therefore be due to this lesion. In addition, among the 285, 634-641. UV-induced base substitutions sequenced by Miller (10), 21 31. Ganesan, A. K., Smith, C. A. & Van Zeeland, A. A. (1981) in of 40 were G-C -> AT transitions at potential (6-4) target DNA Repair: A Laboratory Manual ofResearch Procedures, sites. A study in our laboratory ofall classes oflad mutations eds. Friedberg, E. C. & Hanawalt, P. C. (Dekker, New York), (unpublished observations) revealed that 73 of 177, or 40%, Vol. 1, part A, pp. 89-97. of the UV-induced lacI mutants were G-C -- A-T transitions 32. Franklin, W. A., Lo, K. M. & Haseltine, W. A. (1982) J. Biol. at Chem. 257, 3535-3543. dipyrimidine sites. We therefore suggest that not only is the 33. Gordon, L. K. & Haseltine, W. A. (1982) Radiat. Res. 89, (6-4) pyrimidine-pyrimidone photoproduct a mutagenic le- 99-112. sion, but that it ranks as a major premutagenic lesion in E. 34. Glickman, B. W. (1983) in Induced Mutagenesis: Molecular coli. Mechanisms and Their Implicationsfor Environmental Protec- tion, ed. Lawrence, C. W. (Plenum, New York), Vol. 23, Note Added in Proof. In a lacd shuttle vector irradiated in human cells, pp. the UV mutation 135-170. spectrum closely resembled that in E. coli (40), with 35. Strauss, B., Rabkin, S., Sagher, D. & MQore, P. (1982) the exception that AmiS and Aml6 were recovered at equal Biochimie 64, 829-838. frequency, as in Dcm- E. coli. Since mammalian cells do not 36. Schaaper, R. M., T. A. & methylate CCAGG sequences, (6-4) photoproducts may therefore be Kunkel, Loeb, L. A. (1983) Proc. in Natl. Acad. Sci. USA 80, 487-491. targeting mutations human cells. 37. Kunkel, T. A. (1984) Proc. Natl. Acad. Sci. USA 81, We thank Drs. J. Drake, K. Tindall, T. Kunkel, and D. Fix for their 1494-1498. constructive comments during the preparation of this manuscript. 38. Radman, M. (1974) in Molecular and Environmental Aspects This work was supported by Natural Sciences and Engineering ofMutagenesis, ed. Prakash, L., Sherman, F., Miller, M. W., Research Council (Canada) Grants A2814 and G1598 to B.W.G. Lawrence, C. W. & Taber, H. W. (Thomas, Springfield, IL), pp. 128-142. 1. LeClerc, J. E. & Istock, N. L. (1982) Nature (London) 297, 39. Schaaper, R. M., Glickman, B. W. & Loeb, L. A. (1982) 596-598. Mutat. Res. 106, 1-9. 2. Foster, P. L., Eisenstadt, E. & Cairns, J. (1982) Nature 40. Lebkowski, J. S., Clancy, S., Miller, J. H. & Calos, M. P. (London) 299, 365-367. (1985) Proc. Natl. Acad. Sci. USA 82, 8606-8610. Downloaded by guest on September 24, 2021