Document VJZGVwznagjJewjg0g8BYvy7j

, I. .. MOnCE: This m&fial m a y " # " i ~ ~'*K' ;,Z' ,:?~; ::\',#,,A,,,~.~,~~,,,'I '1'118 .!IllIISAl. IIF ~ ~ l ~ l l . ~ M~~'IIlY~h~llASIlI.lY c l!l!p2 I,, II,,, S~,l,flr t~,,,,.l,,-,,,,qlrnvllrt ~~tv~e,I.I,,ll~:,,rI~I ,~~~,~ ~ . copyright law (Title 17, United States Code). 8-Hydroxyguanine, an Abundant Form of Oxidative DNA Damage, **---Causes G -+ T and A +C ) h b s t h w - EXHIBIT (Received for prililication, July 12, 1991) &*ZQb;r Keith C. C h e n g t , David S. Cahi@!$%&h#k#@ n ~ &' * ~ ~ murag, and Lawrence A. Loeb$ From the SGottstein Memorial Cancer Research Laboratory, lJniv=wton, Seattle. Washington 9819s and the $Biology Division, National Cancer Center Research Institute, Tsukiji 5- I , l , Chuo-ku. Tokyo 104, Japan Mulations caused by oxidative DNA damage may contribute to human disease. A major product of that d a m a g e i s 8- hydroxyguanine (ohRGua).Because of differences in experimental design, the base pairing specificity of oh"G i n v i v o is not completely resolved. Here, oh"dGTP a n d DNA polymerase were used i n two complemen tary bacteriophage plaque color assays to examine the mutagenic specificity of oh"Gua in vivo. The first is n rcvcrsion nsnny I.lint dclccts t i l l Llircc Ringlcbuse substitutions caused by misreading of guanine analogues inserted a t a specific site. oh"Gua at that site g a v e a mutation frequency of 0.7%. Twenty- two of t h e 23 mutRtions w e r e G -+ T substitutions. The second assay, a forward mutation assay, tests the mispairing potential of a n y altered nucleotide 1) d u r i n g incorporation as s u b s t r a t e nucleotide, a n d 2) after multiple incorporations into a single-stranded DNA gap region of M13mp2. Substituting ohHdGTP for dGTP during polymerization produced 16% mutants; two classes of mutations were observed, both caused b y pairing of oh'Gua with A. Seventy-six of 78 mutations w e r e A -+ C substitutions, a n d t w o w e r e G + T substitutions. These assays thus illustrate mutagenic replication of oh"Gua as template causing G -+ T substitutions a n d misincorporation of oh*Gua as subs t r a t e causing A -+ C substitutions, both caused by ohHGua.Amispairs. Cellular DNA is damaged by oxygen free radicals generated during cellular respiration, cell injury, phagocytosis, and exposure to environmental oxidants (Kasai et al., 1986; Fridovich, 1986;Floyd et al., 1986; Klebanoff, 1988; Breimer, 1990). The resultant damage to DNA bases may be a significant source of mutations that lead to cancer and other human pathology (Harman, 1981; Ames, 1983; Pryor, 1986). Because of the multiplicity of DNA modifications produced by oxygen lree radicals (Hutchison, 1981), it has been difficult to establish the frequency and specificity of mutations engendered by individual oxygen-induced DNA lesions. * This work was supported by Fellowship DRG-049 from the Damon Runyon-Walter Winchell Cancer Research Fund (to K. C. C.), National Institutes of Health Outstanding Investigation Grant R35CA-39903 (to L. A. L.), a grant-in-aid from the Ministry of Health and Welfare for a comprehensive 10-year strategy for cancer control (to S. N.),and a grant from the Ministry of Education, Science, and Culture of Japan (to S. N.and H . K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertkement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The guanine analogue 8-hydroxyguanine (oh"Gua)' is an abundant base modification in mammalian DNA whose levels increase with oxidative stress. The amount of ohHGuain cellular DNA is higher in animals with a higher basal metabolic rate (Shigenaga et al., 1989) and is higher in mitochondrial DNA than in nuclear DNA (Richter et al., 1988). Increased levels of oh"Gua are also observed in DNA from yirradiated mouse liver, X-irradiated HeLa cells, and H 0.I rcritrtl .Sdmrm*/kz /yp/tiuturiurtt (Kitsiii c*/ d . , 1!)86).I! Iiiis been estimated that oh%a is formed in human cellular DNA as a by-product of normal metabolic processes at the rate of 178 residues/cell/day (Shigenaga et af., 1989). Of 13 DNA base adducts formed alter exposing purified mammalian chromatin to ionizing radiation-generated free radicals, oh'Gua was the most abundant (Gajewski et al., 1990). The accumulation of oh'Gua is also observed in naked DNA exposed to mutagenic and carcinogenic agents that generate oxygen free radicals (Kasai and Nishimura, 1984; Dizdaroglu, 1985; Floyd et aL, 1986). The importance of ohRGuais underscored by the existence of cellular repair of this lesion, as suggested by the decrease in oh'Gua over time in livers of y-irradiated mice (Kasai et al., 1986). by the urinary excretion of oh"Gua and oh"dG in rodents and man (Shigenaga et al., 1989), and by the finding of a glycosylase/endonuclease activity in Escherichia coli which removes oh'Gua from DNA (Chung et al, 1991). Based upon these findings and the existence of highly sensitive assays for its detection (Floyd et al., 1986), oh'Gua has been the most widely used marker for oxygen free radical damage to DNA (Shigenaga et aL, 1989). Studies of the mutagenic potential of oh'Gua in a variety of experimental systems have yielded different results. Ku- chino et al. (1987) found that oh'Gua not only has the potential to pair with any nucleotide in vitro but may also cause misinsertions at adjacent pyrimidines. Structural studies by Culp et al. (1989) showed that the C6,C8-diketo form of ohXGuapredominates at physiological pH; in syn conformation, this structure is capable of pairing with cytosine, ade. nine, or thymine. Furthermore, the minor, 6-enolate-8-keto form, has the additional potential to pair with guanine (Culp et nl., 1989). I n contrasl, Shibuloni et al. (1991) found that ohXGuapairs with either C or A during in vitro DNA synthesis and does not cause misinsertion a t adjacent bases. Wood et af. (1990) demonstrated the ability of ohHGuato generate G * T.transversions in E. coli. However, the phenotypic screen used, based upon the insertion of the oh'dG in place of the G I The abbreviations used are: oh"Gua, 8-hydroxyguanine (also known as 8-oxo-7-hydroxyguanine,referring to the favored 6,8-diketo tautomer at physiological pH); oh"dG, 8-hydroxydeoxyguanosine; oh"dGTP, 8-hydroxydeoxyguanosine triphosphate; oh'dGMP, 8-hy droxydeoxyguaosine monophosphate; TEAB, triethylammonium bicarbonate: HPLC, high pressure liquid chromatography; Pol, polymerase: HEPES, 4-~2-hydroxyethyl)-l-piperazineethanesulfonaiccid. 166 I I I r P c S I. 7 1; h !I D A at fr 1, hn -ic; he an I - 8-Hydroxyguanine Causes G --j T and A ---t C Substitutions 167 residue i n a 5'-TAG-3' a m b e r codon, was not designed to detect targeted G A transitions, which would yield a different stop codon, 5'-TAA-3'. M o r e recently, Moriya et al. (1991) E. coli Strains and Media-An F' derivative of strain CSH50 (A(pro-lac) thi ora strA/F' (proAB laclq-ZAMfS)), used as plating bacteria, was received from T. Kunkel (NIEHS). Strain NR9161 (mutL21O:Tn10 hsdR- hsdhf' araD A(ara,leu)AlacIPOZY lacUgalK used phosphoramidite synthesis to generate a plasmid con- strA; equivalent to MC1061 mull;), used as the competent calciunl struct with a single-stranded gap containing oh'Gua a t a cells for infectious center plating, was kindly provided by R. Schaaper specific site a n d , using an oligonucleotide hybridization (NIEHS). Strain CJ236 (ung-f dut-f reMf spTI thi-I, pCJ105), screen, also detected G -+ T transversions i n E. coli. However, the number of targeted mutations observed was insufficient to assess t h e possibility that oh*Gua also causes o t h e r muta- tions. used to produce uracil-containing single-stranded M13mp2 and M13G'l DNA, was obtained from Barbara Bachman (E. coli Genetic Stock Center, Yale University). M13C.l was derived from M13mp2 as described (Cheng et al., 1991). 2XYT medium contains, per liter, 16 g of Bacto-Tryptone, 10 g of Racto yeast extract, and 5 g of NaCI. Methods used for t h e insertion of base analogues i n t o DNA P r e p ~ r a t i oo~f oh"dC;TP-The general method of conversion of test constructs often necessitate conditions potentially deg- n~onodeoxyribonucleotideto 5'-triphosphute (Hoard and Ott, 1965) radative to unstable analogues. In both in vitro studies cited above (Kuchino et al., 1987; S h i b u t a n i e t al., 1991) a s well a s the in uiuo studies of Moriya et al. (1991), oh"Gua was introduced into DNA b y phosphoramidite oligonucleotide synthe- was used to prepare oh'dGTP. The rnonodeoxyribonucleotide ohd-5'dGMP was prepared by oxidation of 5'-dGMP (800 mg) in 500 ml of an aqueous solution of ascorbic acid (20 mM), EDTA (20 mM), and FeSO, (2 mM) at 37 'C for 3 h with gentle shaking under air. The pH of the solution was adjusted to 3.0 and mixed with activated charcoal sis, which exposes t h e D N A t o low pH a n d high temperature (5.7 g). The activated charcoal was then packed in a short column during deprotection (Basu a n d Essigmann, 1988). Wood et al. (2.5 X 5 cm), washed with 5 column volumes of water, and eluted (1990) achieved site-specific analogue insertion using enzymatic ligation rather than phosphoramidite synthesis, thereby avoiding deprotection steps. However, a 148-h RNA ligase incubation was required to achieve efficient ligation. Such with 1:l acetone:water (v/v) in 400 ml. 'I'he eluate wns evaporated to dryness, the residue dissolved in 2 ml of water, and the resolubilized nucleotides loaded on a DEAE-Sephadex A-25 column (2.5 X 35 cm). Nucleotides were eluted by a 3-liter linear gradient of 0-0.3 M triethylammonium hicarhonate (TEAR). Elution of oh"dGMP, monitored manipulations m a y have contributed to m u t a t i o n s unrelated by u1)aorliiince i r l 254 nni, wiis tlutccletl ul'ler u large 6'-tl(;MI' peak. to the analogue. The 150-ml oh'dCMP fraction was evapoarted to dryness. Addition We have synthesized oh'dGTP t o allow D N A polymerase to insert t h e analogue into DNA; two independent and complementary bacteriophage assays were used to determine the mutagenic potential of oh*Gua i n E. coli. In the first assay, of water and evaporation was repeated to remove TEAB (yield, 5.8% from starting material). 'I'he phos~lioriiiiidilzc,lid~tIe'orlned I'rum oh"dCM1' and l,l'-car- bonyldiimidazole was reacted with pyrophosphate to yield oh"dGTP (Hoard and Ott. 1965). The products were dissolved in 2 ml of water called t h e M13G*l assay' (Cheng e t al., 1991), oh"Gua is site and loaded on a 2 x 20-cm column of DEAE-Sephadex A-25 pre- specifically incorporated opposite a template C; all three equilibrated with 0.05 M TEAB and eluted with a 500-ml linear noncomplementary single-base substitutions c a n be detected after transfecting a n d replicating t h e DNA in E. coli. I n t h e second, a forward mutation assay, oh"Gua is incorporated into a single-stranded DNA gap; this allowed us to detect multiple gradient of 0.05-0.1 M TEAB, followed by another 200 rnl of 0.1 M TEAB (Fig. 1.4). The pooled oh"dG'I'1' (57 ml), followed by absorbance at 254 nm, was evaporated to dryness. Addition of water and e v ~ p o r d o iwi ere repeated to reinove 'IWAIi. An aliquot representiiig '/x>of this material was injected into an HP1.C column (Beckman &insertions of oh'dGTP which occur during a n in vitro Ultrasphere ODS, 5 pm, 4.6 X 250 mm) and eluted with a solution incorporation step, a s well as to detect miscoding during DNA containing 12.5mM citric acid, 25 mM sodium acetate, 30 mM NaOH, replication of analogue-containing template in uiuo. Results from both assays indicate that d u r i n g DNA replication in E. coli, oh"Cua most frequently codes correctly for C, b u t also has the monospecific mutagenic ability to pair with A to generate transversions at a frequency of about 1%. EXPERIMENTAL PROCEDURES Materials and Enzymes-The four normal deoxynucleotide triphosphatesand dGMP were obtained from Pharmacia LKB Biotechnology Inc. [y-:"PJATP was obtained from Amersham Corp. Oligonucleotides were synthesized and HPLC-purified by Operon Tech- and 10 mM acetic acid (now rate 1 ml/min; Fig. 1B). The oh'dGTP fraction, collected in 1 ml, was evaporated to dryness, the residue dissolved in 0.1 ml of water, and the sample desalted by loading on the same HPLC column prewashed with 30 ml of water, and eluted <I',bwith water. HPLC analysis of the purified product showed that contaminating dGTP (Fig. 1B; retention time 5 min) was completely separated from oh"d'2TP (retention time 8 min). This HPLC purification was repeated (yield 40% from oh'dCMP). The maximal con- Di nologies. The DNA oligomers were 5'-end labeled using T4 polynucleotide kinase (U. S. Biochemical Corp.) and [r-:"P]ATP ............ (Sambrook et al., 1989), with an unlabeled ATP chase, resolved by electrophoresisthrough a 15% denaturing polyacrylamide sequencing pel (Sambrook et al., 1989), and purified by elution from Nensorb 20 columns (Du Pont) with 50% methanol. Uracil-containing single- B O0 64 ,..' 02 stranded UNA was prepared by growing the desired phage strains in 0.0 E, coli strain CJ236 (dut ung), as described (Sambrook et al., 1989). 0 50 100 The uracil-containing DNA was treated with bovine pancreatic Fraclmn number RNase A (type IIA, Sigma) a t 20 pg/ml for 30 min at 45 'C to hydrolyze contaminating RNA that otherwise hybridizes to the M13 template and serves as primers for DNA synthesis (data not shown). DNA grade Sephadex '2-100 was obtained froin Pharmacia and B A C ~ O - Agar from Difco. Polyacrylamide was prepared from 40% 19:l acryl- arnide:bisacrylamidestock from Fisher. Exonuclease-deficient large fragment mutant enzyme D424A of E. coli DNA Pol I (exo- DNA Pol I, Derbyshire et aL, 1988) was kindly sent by C. Joyce (Yale). Native hacteriophageT7 DNA polymerase was obtained from U. S. Biochem- icol Corp. FIG. 1. Synthesis a n d purification of oh"dCTP. oh*dGTP was 'The name refers to the vector's M13mp2 background, and to this synthesized and purified as described under "Experimental Proce- Iieing the first assay we have developed specifically for guanine dures." A, separation of nucleotide triphosphates by DEAE-Sephadex unalogues ("C"'). A-25 column chromatography. B,purilication of oh*dGTP by HPLC. `. 168 8-Hydroxyguanine Causes G -+ T and A + C Substitutions tamination of final product with dGTP was less than 1part in 100,000. UV spectra of the mono- and triphosphates matched those of other syntheses that were characterized further by NMR and direct mass spectral analysis by fast atom bombardment (data not shown; Ikehara et al., 1965; Kasai and Nishimura, 1984). Site-specific Extension of Oligonucleotides-HPLC and gel-purified synthetic 17-nler !i'-d(CCTATTACCCCAGCTGC)-(3c'omplementary to nucleotide positions 6321-6337 of the M13G'l genome; see Cheng el al., 1991, for derivation from M13mp2) was 5` end labeled with [-y-"P]ATP (Sambrook et al., 1989), with a subsequent addition of unlabeled 5 pM ATP and incubation for 15 min a t 37 "C followed by 10 min a t 65 `C. Labeled 17-mer was hybridized to uracil-containing M13G.1 a t a 2.51 primer:template ratio in 300 mM KCI, 100 mM HEPES, pH 7.3. One pg of primed template was incubated for 1h at 30 `C in a 100-pl reaction mixture containing 20 mM Tris-HC1 (pH 7.5). 10 mM MgClp,23 mM KCI, 2 mM dithiothreitol, 1 unit of exoDNA Pol I, and 100 p~ oh'dGTP. Thereafter, each of the four normal deoxynucleoside triphosphates was added to the reaction mixture to a final concentration of 250 p~ followed by incubation for 30 min at 37 `C. Four unita of T7 native DNA polymerase were then added to the reaction mixtures followed by incubation for 20 min a t 37 'C. The reaction was stopped by the addition of EDTA to 15 mM and stored a t -20 `C until used for transformation of competent NR9161 cells. An aliquot of 5-20 pl of the reaction was directly added to 200 pl of competent mutL E. coli strain NR9161 (see below) without further processing. Preparation of Capped M13mp2 DNA-The gapped M13mp2 DNA used was composed of an intact single-stranded circular uracil-con- taining plus (+) strand DNA and a linear minus (-) strand with a 631-base deletion opposite positions 5962-6323 of the Vecbase sequence of M13mp2. This deletion extends from outside the promoter into the amino-terminal portion of the lacZa gene of M13mp2 (to position +145 relative to the start site of transcription). Since this assay scores for decreases or loss of a nonessential gene function (8galactosidase a-complementation), a wide variety of mutations, including single-base substitutions, insertions, and deletions, are detected (Kunkel, 1986).In the gapped DNA assay used here, mutations would be detectable from bases -68 to +145 relative to the start site of transcription of the lacZn gene. The incomplete strand was derived from M13mp18, which is essentially the same as M13mp2 except for a restriction enzyme polylinker at the EcoRI site in the deleted region. Since the deleted region is very small compared with full-length DNA, we used an M13mp18 construct containing a 1.7-kilobase insert in the deleted region to increase agarose gel resolution between single cut and fully cut plasmid DNA. The desired double-stranded 6835base pair PuulI restriction endonuclease fragment was combined with uracil-containing single-stranded M13mp2 DNA to produce gapped DNA. Annealing was accomplished by dialyzing the combined DNAs in progressive dilutions of formamide (Lundquist and Olivera, 1982). The desired Purr11 fragment and gapped DNAs were purified from 0.8% preparative agarose gels by electroelution (Sambrook et af., 1989)without exposure to potentially mutagenic UV light or ethidium bromide. T o do so, 0.8-cm strips were cut from each side of the preparative gels and stained in 0.5 pg/ml ethidium bromide. The desired bands were visualized in the stained strips and marked by cuts above and below the bands; the corresponding bands were then excised from the unstained preparative gels using the cuts for alignment. Gap Filling in the facZa Region of MZ3mpZ-The reaction mixtures in the gap-filling assay contained 400 ng of gapped DNA (above),20 mM Tris-HCI (pH 7.5). 10 mM MgCI,, 2 mM dithiothreitol, 0.2 units of exo- DNA Pol I, and the indicated concentrations of dNTPs (see Table 11). The reactions were incubated for 8 min at 37C and stopped by adding EDTA to 13 mM. One-pl aliquots or 1 pl of 1:lO dilutions were used to transform 200 pl of competent E. coli (below). Competent Cell Preparation, Plating, and Ploque PurificationCompetent NR9161 cells were prepared using CaCI,, and transfection and plating were performed as described previously (McBride et al., 1991).Multiple transformations and platings yielded similar mutation frequencies. In the M13G'l assay, dark blue plaques representing revertants were scored, purified, and sequenced. In the gap filling assay, light blue and white mutant plaques were restreaked on bacterial lawns for purification but otherwise processed identically to the M13G'l assay. RESULTS DNA polymerase-mediated analogue insertion was used in two different bacteriophage assays to determine the mutagenic potential of oh'Gua. O u r assays differ from t h e earlier ones in requiring neither oligonucleotide synthesis nor lengthy ligase incuhations. T h e synthesis of o h " d C T P (see "Experimental Procedures") allowed t h e rapid DNA polymerase-mediated introduction of oh'Gua into bacteriophage constructs. In addition, both assays utilize a uracil-containing template s t r a n d to bias for replication of t h e analogue-containing s t r a n d lacking uracil (Kunkel, 1985; Reid et al., 1990). T h e first assay, the M13G*1 assay, detects all three single base substitutions opposite a single site-specifically incorporated guanine analogue (Figs. 2 and 3A). T h e second scores for forward mutations after t h e insertion of modified bases at multiple positions within a defined region of a DNA template (Fig. 3B). Site-specific Mutagenesis by oh'Gua-To determine t h e coding specificity of o h R G u aresidues i n DNA during replication i n E. coli we first used a reversion assay, t h e M13G*1 assay (Figs. 2 and 3A). T h e assay is based upon restoration of @- galactosidase activity by any single-base substitution at the target site, resulting in a change i n plaque color from white to blue (Fig. 2). Site-specific incorporation of ohRGua in M13G*1 was accomplished in a series of reactions (Fig. 3A). The uracil-containing single-stranded circular M13G*1 template DNA was first hybridized to a n oligonucleotide such t h a t the n e x t base t o be added onto t h e 3' primer terminus was opposite the target cytosine a t position 141. Site-specific insertion of ohRGuaopposite C141 was accomplished using ohRdGTPa s the sole nucleotide precursor for exo- DNA Pol I. During this first step, more than 90% of t h e 17-mer primer w a s elongated to a n 18-mer, with no further extension observed; 10% of t h e primer remained unextended (data not shown). The nucleotide analogue was then sealed into the nascent strand by t h e subsequent addition of all four normal dNTPs a t high concentration followed by T 7 DNA polymerase to generate fully double-stranded product. Native T 7 DNA polymerase w a s chosen because it is highly processive and exhibits little strand displacement during DNA synthesis (Lechner a n d Richardson, 1983), i n contrast to t h e modified versions of T 7 polymerase ( T a b o r and Richardson, 1989 "Sequenase"). T h e DNA product was directly transformed into competent E. coli to minimize delay a n d manipulations between analogue insertion a n d transformation. In control experiments (not shown), we determined that further exten- sion by T 7 DNA polymerase caused a n increase in transformation efficiency by at least 2 orders of magnitude. Revertant blue plaques were scored, purified, a n d sequenced (Fig. 3 A ) . T h e mutation frequency obtained after insertion of oh"Cue a t C141 was 32-fold greater t h a n t h a t obtained i n controls; 22 of t h e 2 3 mutations were G + T transversions (Table I). In contrast, with dG at position 141, eight of t h e nine mutations were G A transitions, a n d n o n e were G --f T transversions -3 ~ 1 3 0 ' 1 5' G-5 oplpr F 3' white 141 FIG. 2. T h e M13G11 site-specific reversion assay for the determination of mutagenic spectrum of guanosine analogues. All three single-base substitutions a t the site of analogue insertion at Cys"' are detected by a change in plaque color. 8-Hydroxyguanine Causes G --.f T and A 4 C Substitutions 169 A M13G'l Site-specific Reversion Assay B M13mp2 Gapped DNA Forward Mutation Assay M13G'l (whlte) I1. oh'dGTP. KO-DNA POI I 2. dATP. dCTP. d R P . dGTP 3.17 DNA polymerase 1dATP. dCTP. d R P (5wY) E x 6 DNA POI I I Transfect. I Rc. 3. Two in uiuo assays for oh"Gua mutagenesis. Panel A, the M13G*1 assay. Uracil-containing plus-strand M13CC1 was primed with a 17-mer such that the next template nucleotide was C"'. oh%ua was then inserted opposite C"l using DNA polymerase and oh'dGTP as the only nucleotide substrate. After analogue insertion, all four normal dNTPs and finally native T 7 DNA polymerase were added to complete extension. The copied circle was then transfected into competent E. coli and plated in the presence of isopropyl I-thio-8-D-galactopyranosiadend x-Gal for detection of blue rever- tant plaques. The revertants were sequenced to determine which of the three possible single-base substitutions gave restoration of 8- galactosidase activity. Panel B, insertion of oh'Gua into gapped h113mp2 DNA. Gapped DNA containing uracil in the plus strand was prepared and reactions carried out as described under "Experi- mental Procedures." Incorporation of oh"Gua into the single-stranded gap was catalyzed by exo- DNA Pol I in the presence of oh"dGTP, dA'I'P, dCTP, and dTTP. After nucleotide incorporation, the DNA was transfected into competent E. coli, and mutant DNA from white and light blue plaques was sequenced to localize the inactivating mutations. In both A and B , arrowheads outside the circles represent 3' DNA termini. In the template plus strand DNAs about 10%of the thymines have been replaced by uracil (Sagher and Strauss, 1983). TABLIE Site-specific mutagenesis by oh%ua In the normal G control, the guanine base was positioned at the target site by hybridization of an 18-mer that contained a 3'-terminal dG. Copying reactions, transfections, platings, mutant purification, and sequencing were performed as outlined in Fig. 2B and detailed under "Experimental Procedures.'' Base at No. of 10' x position plaques Om Mutation No. Mutation 141 screened Mutants frequency oh'Gua G ua 3,010 37,000 0.76 0.024 G -+T G-C G-A G +T G+C G-rA 22 0 1 0 1 0 72 ~3 -3 ~0.3 -0.3 2 ('roble I). A combination of experiments, yielding a total of 38,000 plaques, indicates that the background frequency of G -+ T substitutions at this position in the M13C*1 assay is ahout 0.13 X lo-', more than 500-fold less than the frequency of 72 x IO-' caused by oh"Gua. We sequenced about 175 nucleotides in each of the 23 oh"dGTP-induced mutations; no untargeted nucleotide sequence alterations were observed. In other experiments, SOS induction did not affect the frequency or spectrum of mutations (data not shown). Mutagenic Spectrum of oh*Gua in a Forward Mutation Assay-To test the mispairing potential of ohnCuain multiple sequence contexts and to examine the potential ability of ohHGuato cause insertions, deletions, or mutations of adjacent bases, we have also scored forward mutations after analogue insertion at multiple sites in a defined region of the lac& gene of M13mp2. Gapped DNA was used to 1) incorporate analogue over a defined region of DNA; 2) increase transformation efficiency of product DNA without further polymerase incul)uLioriu; uncl 3) ~irotcclphage genes essenliul for virul viability by making those regions double stranded; polymerization into the double-stranded DNA regions would then require strand displacement synthesis. By using unbalanced concentrations of nucleotides one can favor the misincorporation of noncomplementary nucleotides (Zakour et ~ l . 1, 984). Modified nucleoside triphosphates can be used for DNA polymerase-mediated incorporation into DNA (Eadie et al., 1984; Preston et al., 1986). These two principles, together with the use of a 3' 5' exonucleasedeficient DNA polymerase, exo- DNA Pol I (Derbyshire et al., 1988),and biasing for replication of analogue-containing strand (Kunkel, 1985; Reid et al., 1990), were combined here in a forward mutation bacteriophage assay that examines 1) base pairing potential of oh*dCTP as a substrate, and 2) miscoding potential of analogue as template at multiple sites during DNA replication in E. coli (Fig. 38). After transfection into E. coli, lac20 mutants are identified by their white or light blue plaque color against a background of' nonmutant dark blue plaques. We first examined the dependence of transformation and mutation frequencies on the ratio of ohHdGTPto normal nucleotides (Table 11). Substitution of oh"dGTP for dGTP in the extension reactions resulted in a decrease in relative transformation efficiency to that obtained for unextended DNA (Table 11). This decrease is consistent with poor elongation. Increasing the concentration of oh"dGTP increased the frequency of mutations. This concentration dependence suggests that the mutations were caused by the presence of oh"dGTP rather than the absence of dGTP. When the concentration of oh'dGTP was 100-fold higher than dATP, dCTP, and dTTP, the mutation frequency was 16%. Of 61 mutants sequenced from this reaction, 47 contained single mutations, and 14 contained two to four mutations each. The mutations showed striking specificity and localization (Fig. 4). Among the 78 base substitutions observed in the gapped DNA forward mutation assay, 76 were A . T --.,C. G transversions opposite plus (+) strand template adenines within the gap region. Two mutations represented G . C -+ T . A transversions at positions where the plus strand base was cytosine; these occurred in separate mutants, each containing multiple mutations. No mutations were observed in bases adjacent to putative ohHdCMPinsertion sites, and no insertions or deletions were detected. The number of mutations was largest near the 3' primer end of the gap (the right side of the gap in Fig. 3B) and decreased with distance; the farthest observed was 71 nucleotides from the 3' end of the gap even though the gap continued for another 290 nucleotides (Fig. 4). This distribution of mutations is consistent with poor elongation from 3'-oh8dG termini suggested by the transformation data (see above). -170 8-Hj.droxjguanine Comes G -t T and A C Substitutions T.BLE 11 Transformation e//iciencim and mutation /requenciea in the M13mp2 zap a9.qay Reactions and transfections were done as detailed under "Experimental Procedures" and outlined in Fig. 28. The percentages of each type of mutation were calculated on the basis of the total of 78 single-base substitutions among the 61 sequenced mutants from the reaction containing 500 p M oh'dCTP. The relative efficiency of transformation refers to the number of plaque-forming unitslng of DNA, divided by the value obtained for the gapped DNA reaction to which no nucleotides were added. Relative No. of - [Deoxynucleotide triphosphate] efficiency of Mutants plaques 5 Mutations transformation screened Mutants None 5 pM MTP, dCTP, dTTP, dGTP 5 p M MTP, dCTP, dTTP; 5 PM oh'dGTP 5 fiM dATP, dCTP. dTTP; 50 PM ohRdGTP 5 PM dATP, dCTP, d'ITP; 500 phl ol1"dGTP 1 10 1 1 I 0 319 c n . 3 ~~ 0 1,222 c0:os 0 417 co.2 2 398 0.5 61 385 1G Not sequenced. .tw *' JGapregbn FIG. 4. Spectrum of ohaGua-induced mutations. A total of 61 mutants obtained from the gappe, 0 IA assay (Fig. 2B) were sequenced. Some of the mutants contained more than one mutation, yielding a total o 78 mutations, all single-base substitutions. These mutations (plus strand changes shown) are indicated above he wild-type sequence shown here, which represents the template plus (+) strand in the hcZu gene; the numbers refer to position from the start of lacZ transcription. Although the gap is 361 nucleotides long (further than the sequence shown here), all of the mutations were present within the first 71 nucleotides of the start of the gap. DISCUSSION The nonspecific coding of ohRGuaand loss of coding specificity at adjacent pyrimidines, observed by Kuchino et al. (1987) in vitro, and the promiscuous base pairing potential of ohRGuasuggested by Culp et a!. (1989) make it important to establish whether a similarly wide spectrum of mutations may also be induced in vivo. Studies in E. coli by Wood et ai. (1990) and Moriya et al. (1991), which demonstrate targeted G +T transversions, partially answer this question. To ascertain the mutagenic potential of ohXGuaduring replication of oh"Guacontaining DNA in uiuo we have used two genetic assays in which the placement of ohRGuain double-stranded DNA was accomplished using oh'dGTP as substrate for DNA polymerase (Fig. 3). In a site-specific assay, the M13G'l assay (Figs. 2 and 3 A ) we observed that a single oh"Gua raised the mutation frequency more than 30-fold above background to 0.7%; 22 of 23 mutations were G + T transversions (Table I). No G +T transversions appeared among nine control mutants. in the second assay, utilizing a double-stranded DNA with a single-stranded gap (Fig. 3B), mutations were scored after polymerase-mediated incorporation of analogue into the minus strand of the promoter/amino-terminal region of the lacZn gene. To bias for replication of the analogue-containing strand during replication in E. coli, uracil is present in the template (+) strand but not in the gapped (-) strand (Kunkel, 1985; Reid et al., 1990). In this forward mutation assay, insertions, deletions, and single base substitutions are detectable, as are multiple mutations in single mutants. We observed only two categories of single-base substitutions, however. In the first category, mutagenic pairing of the analogue with template adenine occurred in vitro during incorporation of analogue as incoming base. In the second category of mutants, mutagenic pairing of A with template oh"Gua occurred during in vivo DNA replication after correct insertion in multiple sequence contexts. The first and most abundant category of ohRGuamutants in the gapped DNA assay was at plus strand A positions. Here, ohRGua as an incoming base paired with template adenine during the in vitro DNA replication step. The insertion of ohRGuain the new minus strand was likely followed by pairing of the ohRGuawith C during replication in E. coli to generate A +C transversions (Table I1 and Fig. 4). Other sources of these mutations are unlikely. Pairing between the new minus strand ohRGuawith dATP during E. coli replication would restore the original sequence and not register as a mritnlion. Repair of oh"C.iin to G nppcnrs unlikely 1)ecnriseof resistance of oh"Gua to excision by oh'Gua DNA glycosylase when paired with A (Tchou et al., 1991), as well as to the resistance of 3'-terminal A to 3` --.) 5' exonucleolytic proofreading by DNA polymerase (Shibutani et al., 1991). The second type of mutation in the gapped DNA assay is analogous to those occurring in the M13G*1 assay; these occurred a t plus strand C positions. Here, oh'Gua was first inserted in uitro in the minus strand across from plus strand C positions. During DNA replication in E. coli the minus strand ohRGuamiscoded for A, producing G + T transversions. From the numbers and types of mutations within the gap region, one may derive an in vivo estimate of the average frequency of mutations during replication of oh'Gua-contain. ing DNA. Consider a hypothetical mutant (below) with a single mutation M opposite template A, in which 1) sequence between the mutation and the 3' end of the original single stranded DNA gap is shown; 2) the lower strand is the plus strand; and 3) X in the plus strand represents any nucleotide except C. .3`. . M X X M L W X X X X X X X X ~ .. .5' 5`. . . A ~ ~ X X. . 3 ' X ~ ~ , During synthesis of the upper, minus strand, DNA polymerase 8 - H y d r o x y g i i a n i n e Causes C; +? ' a n d A -+C S u b s t i t u t i o n s 171 encountered three template Cs as it proceeded from the start dGTP on poly(dA) by E. coli DNA Pol I11 in uitro (Akiyama of the gap on the right to the mutation site M on the left. et al., 1989). The model for A.G mispairing of Topol and Since normal dGTP was not present during DNA synthesis, Fresco (1976) and work by Bhatnagar et al. (1991) with oh'Gua was presumably inserted at each of the plus strand nucleotide analogues suggested that the enzyme might prefer template C sites. In this example, oh"Gua coded for C during a syn tautomer of dGTP, which is the predominant form of subsequent DNA replication or was repaired, since no muta- ohRdG(Culp et al., 1989). In fact, H. Maki and M. Sekiguchi3 tions were observed at those template C sites. In our experi- have recently found that the MutT protein hydrolyzes ments we totaled the number of template Cs between the oh'dCTP to monophosphate much more efficiently than mutation and the start of the gap in each mutant; for m u m & dGTP. Therefme, thisspecificity net c ! y eccoanb f ~ trhe with more than one mutation, we counted from the mutation mutagenic specificity of mutT mutators but also accounts for farthest to the left as drawn above. The sum of all putative the appearance of ohXdGas nucleoside in urine (Shigenaga et oh"Guaincorporations opposite template Cs in the 61 mutants d., 1989); mutM/fpg-1 glycosylase activity, in contrast, re- (Table 11) was 338. Since two template C mutations were leases free base (Bailly et d.,1989). The results from the observed,the calculated mutation rate is 2/338, or about 0.6%. gapped DNA assay thus implicate oh'dGTP as a possible This frequency is similar to the mutation frequency of 0.7% source of oxygen-induced A .T +C.G transversions. observed in the M13G*1 assay. The low mutagenic potential of oh'Gua may be caused by The gapped DNA assay (Fig. 3B) can be applied to adenine, repair of oh"Gua (Tchou et al., 1991) or to low miscoding cytosine, thymine, or guanine deoxynucleoside triphosphate potential, or both. This low mutagenic potential allows greater analogues. This approach is dependent upon the ability of the survival in the face of the putatively high steady-state number polymerase both to utilize the modified deoxynucleoside tri- of oh'Gua residues in cellular DNA (about 105/cell; Fraga et phosphate as a substrate and then to extend DNA from 3' al., 1990). The presence of this modified base in cellular DNA termini containing the modified base. Since mutations in suggests that excisional repair of oh'G is not complete. Hence, genes necessary for phage production can prevent the detec- ohRGuamay still play a major role in the generation of oxygen- tion of concurrent mutations in the lacZa target gene, we induced G . C -+ T - A transversion mutations. limited incorporation to the region of the nonessential target The mutagenic specificity of oh"Gua provides a potential gene by the use of gapped DNA, whose double-stranded region tag for assessing the role of oxygen free radical mutagenesis covers essential phage genes. in human cancer. I t is of interest that the spectrum of p53 The data from the two assays used here suggest that in E. tumor suppressor gene mutations includes G. C -+ T - A trans- coli, ohRGuais nonmutagenic about 99% of the time but a t a versions in about half of non-small cell carcinomas of the 4%frequency, yields G .C T.A transversion mutations lung and nearly three-quarters of primary liver carcinomas by base pairing with A. The G .C + ".'A transversions ob- (Hollstein et al., 1991). In primary liver carcinoma, which is served here were unlikely to be the result of depurination associated with hepatitis B virus infection, G . C + T . A I (Schaaper et al., 1983) or of copying past bulky base lesions (Straws et ol., 1982). Mutagenesis via depurination is SOS transversions are the predominant p53 gene mutation. Oxygen free radicals, which are a side product of chronic inflammation 1 t dependent; the mutations obtained here were SOS independent.This analogue does not appear to act as a bulky derivative since it can be readily inserted and copied by DNA polymer- ase. Moreover, the base pairing observed is also consistent with NMR studies showing that ohndG (anti) stably pairs with either C ( a n t i ) (Oda et al., 1991) or, with A (anti)in syn conformation (Kouchakdjian et d., 1991). Our results are entirely consistent with the in uitro results of Shibutani et al. (Cerruti and Trump, 1991), may be produced by the many inflammatory cells present in the liver during chronic hepatitis B virus infection (Cotran et al., 1989). If these free radicals contribute to G - C 4 T . A transversions by the production of oh"Gua in liver DNA, therapies designed to reduce damage by oxygen free radicals (Ames, 1983; Imlay and Linn, 1988)during chronic hepatitis would be predicted to delay the onset of primary liver cancer. (1991),the in uiuo results of Wood et al. (1990), and the poor repair of 0h'Gua.A pairs by E. coli oh'Gua-DNA glycosylase Acknowkdgments-We thank Drs. A. Blank, M. Fry, and T. Reid for comments on the manuscript;A. Lee and T. Nguyen for excellent reported by Tchou et al. (1991). The relevance of this lesion technical assistance; Drs.T. Kunkel. R. Schaaper, and B. Bachman and its associated mutagenesis in vivo is also supported by for E. coli strains; and M. Bohidar and K. Carroll for their excellent 1heG.C 4T.A transversion mutator phenotype of a probable graphics work. mutant in the E. coli oh"Gua repair enzyme, mutM (Cabrera et a l , 1988). 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