Proc. NatL Acad. Sci. USA Vol. 80, pp. 4446-4449, July 1983 Genetics Targeted mutation at cytosine-containing pyrimidine dimers: Studies of Escherichia coli B/r with acetophenone and 313-nm light (suppressor mutation/sensitized irradiation/SOS induction/transdimer synthesis) DOUGLAS FIX AND RICHARD BOCKRATH Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana 46223 Communicated by Richard B. Setlow, April 7, 1983 ABSTRACT We have tested the two-event model for UV mu- dexing signal and a premutational photoproduct that targets the tagenesis producing class 2 suppressor mutations at glutamine tRNA mutation (12-14). While the first of these could be any lesion genes in Escherichia coli. In the model used, the induction/in- that disrupts DNA synthesis to stimulate SOS induction or in- dexing lesion is any type of pyrimidine dimer and the premuta- dex critical overlapping daughter-strand gaps (15), the targeting tional photoproduct at the target site is a cytosine-containing di- photoproduct could be unique. For example, the transitions mer. Specific mutation-frequency responses were analyzed under accounting for the three examples of class 2 suppressor mu- conditions in which the ratio of thymine-thymine dimers to cy- tation affect cytosine residues to the 3' side of thymine residues tosine-containing dimers was modified by using 313-nm light and in the DNA target sequences. Therefore, the possibility of mu- 0.0%, 0.1%, or 0.2% acetophenone. Changes observed in the pro- duction of class 2 suppressor mutations were consistent with the tation at a thymine-cytosine (TC) pyrimidine dimer as the pre- model and suggested that the G-C -- AT transitions responsible mutational photoproduct has been proposed (16-18). This was for class 2 suppressor mutations are targeted by cytosine-con- supported by results showing cytosine-containing dimers to be taining pyrimidine dimers at the mutational sites. a likely premutational photoproduct for class 2 suppressor mu- tations (19). Cells mutagenized with 254-nm light were held in Mutagenesis in bacteria is now known to have considerable buffer at an elevated temperature and irradiated with 405-nm specificity. This is seen in distinct nonrandom or contrasting light to cause photoenzymatic monomerization of pyrimidine patterns of induced DNA alterations by mutagenic agents and dimers. This irradiation eliminated revertants except in cells suggests targeted phenomena (1, 2). With UV mutagenesis of lacking uracil-DNA glycosylase activity. When repair of uracil the lad gene, a major portion of the data is consistent with tar- residues in DNA was absent, class 2 suppressor mutations spe- geting at cytosine-containing pyrimidine dimers or pyrimidine- cifically became refractory to the 405-nm irradiation. This im- pyrimidine (64) photoproducts (3), but additional features are plicated a TC premutational photoproduct that could degrade clearly important to explain the "hot spots" preferred for these to a thymine-uracil dimer and be repaired by photoenzymatic mutations among all possible sites (4). Reversion mutation also monomerization to establish the cytosine to thymine (uracil) may be used to study specific DNA alterations if nonsense-de- transition responsible for class 2 suppressor mutation. fective auxotrophs are mutagenized. When UV-induced re- We tested the two-event mutagenesis model by assuming vertants of a nonsense-defective strain are selected on semi-en- that there is a qualitative difference in the type of photoproduct riched medium, a pronounced preference for the production of required for the two separate events. Irradiation with 313-nm suppressor mutations at glutamine tRNA genes is observed (5, light alone produces approximately two cytosine-containing di- 6). These class 2 suppressor mutations (7) indicate specific base- mers for every one thymine-thymine (TT) pyrimidine dimer [in pair changes and large numbers are easily distinguished. E. coli (20) or animal cells (R. B. Setlow, personal communi- Therefore, it is possible to associate the frequency of these spe- cation)]. However, in the presence of the photosensitizer ace- cific alterations quantitatively with different mutagenizing ex- tophenone, which transfers triplet state energy to thymine suf- periences. ficient only for the formation of TT dimers (21), the distri- Reversion of nonsense-defective auxotrophic cells can occur bution of pyrimidine dimers can be sharply altered to favor the at several sites in the bacterial genome-e.g., suppressor mu- formation of TT dimers (22, 23). We used this procedure to tations at different tRNA loci and back mutation in the non- introduce various distributions of pyrimidine dimers and de- sense-defective gene (7). The two species of glutamine tRNA termined specific mutation-frequency responses. For the three in Escherichia coli recognize the codons CAA and CAG and are examples of class 2 suppressor mutation, the responses changed encoded by tandem duplicates of the genes glnl and gln2, re- in a manner consistent with the model that induction/indexing spectively (8). Class 2 suppressor mutations result from G'C is by any type of dimer but the premutational photoproduct at A-T transitions at the distal or proximal ends of the anticodon the target site is specifically a cytosine-containing pyrimidine sites in these genes producing de novo ochre suppressor mu- dimer. tations (glnl -- glnl°) and de novo amber suppressor mutations (gln2 -- gln2a) (9, 10) or converted amber-to-ochre suppressor MATERUILS AND METHODS mutations (gln2a gln20) (11). The process by which these suppressor mutations arise may Excision-repair-defective strains of E. coli B/r, WU-11 and WU, require two types of DNA damaging events, an induction/in- were used (24). Cells were grown and irradiated with 313-nm radiation (2.5 W/m2) from a 1,000-W Hg/Xe lamp (19) filtered The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertise- Abbreviations: TT, thymine-thymine pyrimidine dimer; TC, thymine- ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact. cytosine pyrimidine dimer. 4446 Downloaded by guest on September 24, 2021 Genetics: Fix and Bockrath Proc. Natl. Acad. Sci. USA 80 (1983) 4447 through redistilled pyridine (25% in toluene) and by broad and mutations. All responses displayed fluence-squared increases narrow band pass filters (Oriel 5180 and G-521-3130). Just be- and were clearly enhanced by the addition of 0.1% or 0.2% ace- fore irradiation, a 10% (vol/vol) solution of acetophenone (East- tophenone to the irradiation buffer. An example is shown in man Kodak) in dimethyl sulfoxide (22) was diluted into the cell Fig. 2 for the conversion of class 2 suppressor mutations in WU- suspensions to the final concentrations indicated. After irra- 11 in the absence or presence of 0.1% acetophenone. Again, a diation, survival and mutagenesis were assayed on semi-en- dose-adjustment factor could be used to superimpose the data riched media (Difco nutrient broth, 0.2 mg/ml) as described (filled symbols labeled 1.4 in Fig. 2) but the dose-adjustment (24). Specific types of revertants were distinguished by their factor for mutation was not the same as that for survival. ability to propagate nonsense mutants of bacteriophage T4 (NG A selective increase in particular types of dimers by aceto- 75, NG 273, and PS 205) (18, 19). Tyr' reversion mutations pro- phenone could be expected to enhance inactivation and mu- duced by UV in WU-11 are primarily either back mutations of tation differently if survival were a function of any type of di- the UAA codon in the tyrosine gene (tyr- tyr') or class 2 mer and mutation depended, at least in part, on a particular ochre suppressor mutations. Since this strain carries a class 2 type of dimer. All of the data suggested dissimilar dose-ad- amber suppressor mutation (supE or gln2a) that suppresses a leu justment factors for survival and for mutation and thus that in- (UAG) mutation, the induced class 2 ochre suppressor muta- activation and mutagenesis resulted from different sets of pho- tions are either de novo suppressor mutations (glnl -- glnlI) toproducts. To determine whether the differential responses or converted suppressor mutations (gln2a -4 gln20). Strain WU might be consistent with distinct models, simple mathematical does not carry the class 2 amber suppressor mutation and this functions for survival and mutation-frequency responses were allows Leu+ reversion to be studied in addition to Tyr' rever- considered and tested with the data. sion. Leu+ revertants are primarily either back mutations (leu- -* leu+) or de novo class 2 amber suppressor mutations (gln2 ANALYSIS gln2a). Lethality and mutagenesis produced by 313-nm light with or RESULTS without acetophenone are approximately 95% or greater photo- Colony-forming ability of E. coli WU and WU-11 was more ef- reversible (22, 23). This indicates that pyrimidine dimers are ficiently inactivated by 313-nm light in the presence of 0.1% the principal cause of lethality by this irradiation. For mutation or 0.2% acetophenone. The survival curves were superimpos- involving two types of damaging events, photoreversibility in- able if dose-adjustment factors were used (2.2 for 0.1%, 3.3 for dicates only that at least one type is a pyrimidine dimer (15). 0.2%). These factors were derived from the exponential por- Nevertheless, in the following models, survival and mutation tions of the inactivation curves and were the numerical values frequencies are described in terms of pyrimidine dimers. This by which the fluence scales (abscissae) could be adjusted so that is considered further in the Discussion. survival data without acetophenone became congruent with We assume survival after 313-nm irradiation follows some survival data in the presence of acetophenone. An example is function S of the occurrence of dimers (t + c), where t rep- shown in Fig.