Proc. Natl Acad. Sci. USA Vol. 80, pp. 4884-4888, August 1983 Biochemistry

Eseherichia coli polymerase I can use 02-methyldeoxythymidine or 04-methyldeoxythymidine in place of deoxythymidine in primed poly(dA-dT)poly(dA-dT) synthesis (fidelity/analogue incorporation/alkylation/helix stability/terminal nucleotidyltransferase) B. SINGER, J. SAGI*, AND J. T. KUWMIEREKt Laboratory of Chemical Biodynamics and Space Sciences Laboratory, University of California, Berkeley, California 94720 Communicated by Heinz Fraenkel-Conrat, April 29, 1983 ABSTRACT O2- and 04-alkyldeoxythymidine are among the given on the possible structural alterations in either the tem- four O-alkyl base-modified derivatives produced by the reaction plate or the resulting polymer. The derivatives studied in this of N-nitroso alkylating agents with nucleic acids in vitro and in way include 06-methyldeoxyguanosine (7), 06-ethyldeoxygua- vivo. We find.that both O2- and 04-methyl-dTTP can substitute nosine (8), and 3-methyl- and 1,N6-ethenodeoxycytidine (9). In for dTTP in alternating poly(dA-dT)-primed DNA synthesis. Up the case of the best-studied derivatives, 06-alkyldeoxyguano- to 22% of the pyrimidines in the newly synthesized polymer were sine, both Abbott et aL (10) and Miller et al. (8) report that the found by HPLC analysis to be O-methyldeoxythymidine. Little triphosphate was not incorporated by pol I and, in fact, ap- polymer synthesis was observed in the absence of dTTP. How- peared to be an inhibitor of normal synthesis. ever, the O-methyl-dTTPs did not inhibit polymerization of dATP like and dTTP. Polymers containing O2_ or 04-methyldeoxythymidine The 0-alkylated thymidines are, 06-alkyldeoxyguano- were obtained in good yield, retaining the secondary structure of sine, products of N-nitroso carcinogen reaction with nucleic acids alternating poly(dA-dT). This was shown by the data for thermal (2, 11). Unlike 06-alkyldeoxyguanosine, they are not rapidly transition under different conditions. In contrast, poly(dA-dT)- repaired in vivo (12, 13). In the present investigation, exper- poly(dA-dT) methylated or ethylated to less than 4% total modi- iments were designed to test whether 02- and 04-methyl dTTP fication by alkylnitrosoureas had a distinctly less stable structure. (02_ and 04-Me-dTTP) could substitute for dTTP in pol I-di- Neither O2- nor 04-methyldeoxythymidine can form more than rected synthesis and, if so, whether their presence altered the one hydrogen bond with adenosine. The unchanged secondary structure or template properties of alternating poly(dA-dT). structure of polymers containing these modified thymidines in- dicates that stacking interactions must play a major role in helix MATERIALS AND METHODS stabilization. O-Alkyldeoxythymidine may be formed by N-nitroso All enzymes were commercial products (P-L Biochemicals and carcinogens that react intracellularly. We have shown that the tri- Bethesda Research Laboratories) except for purified E. coli DNA phosphates can be utilized by Escherichia coli DNA polymerase polymerase I, which was kindly given by L. A. Loeb. Deoxynu- I as dTTP. The incorporated 04-methyl-dT causes misincorpora- cleoside triphosphates and alternating poly(dA-dT) were from tion of G, both in transcription and synthesis. When 02-methyl- P-L Biochemicals. dT is present, less, but definite, misincorporation results. Preparation of O2- and 04-Me-dTTP. O2- and 04-methylde- oxythymidine (02- and 04-Me-dT) were prepared from the iso- The structural and mutagenic effects of many alkyl derivatives propyl derivatives by exchange of the isopropyl group by so- formed by carcinogens in vivo have been investigated in vitro dium methoxylate. The isopropyl derivatives were used because in various ways. Substitution of a noncomplementary nucleo- the reaction conditions led to a high yield of the o2_ and 04- tide has been used as a test of whether the modification causes isopropyldeoxythymidines without significant reaction at the transcriptional errors and whether it changes the ability to form N-3 position. Isopropylation of the oxygens was achieved by a normal base pair (1-3). There is little information on the use with 5 of modified deoxynucleotide triphosphates (dNTPs) in primed reaction of a suspension of 10 mmol of deoxythymidine with g of silver oxide and 10 ml of isopropyl bromide in 100 ml of DNA synthesis using DNA polymerases, particularly those isopropanol. After 2 days at 37°C, additional silver oxide (2.5 high fidelity resulting from their associated proofreading func- g) and isopropyl bromide (5 ml) were added and the reaction tion (4). It has been reported that N6-methyl-dATP can replace on cel- dATP in Escherichia coli DNA polymerase I (pol I)-catalyzed was continued for several hours until chromatography synthesis of the alternating copolymer poly(dA-dT) (5). How- lulose thin-layer plates developed in butanol/ethanol/H20 A-T base (80:10:25) showed no unreacted deoxythymidine. Separation ever, this derivative has the ability to form a normal of o2- and 04-isopropylthymidine was on preparative silica gel pair because the methyl group is oriented anti to the Watson- plates developed with chloroform/isopropanol (80:20). The Rf Crick side when the triphosphate is incorporated. Similarly, 5- of 02-isopropyldeoxythymidine (mp 175-177°C) was 0.27 and substituted pyrimidine dNTPs can also be used by pol I because that of 04-isopropyldeoxythymidine (mp 99-101°C), 0.45. The the modification does not interfere with base pairing (6). Both Carcinogen-modified deoxynucleotides have been incorpo- yields were 25% o2- and 30% 04-isopropyldeoxythymidine. rated into random single-stranded polymers by using terminal derivatives were crystallized using isopropanol for 02-isopro- or modifications have been deoxynucleotidyltransferase specific Abbreviations: pol I, DNA polymerase I; o2- and 04-Me-dTTP, o2- and introduced. Such polymers were transcribed or replicated to 04-methyldeoxythymidine triphosphate; o2_ and 04-Me-dT, o2_ and ascertain whether misincorporations occur, but no data were 04-methyldeoxythymidine; tm, melting temperature. * Present address: Central Research Institute for Chemistry of the Hun- The publication costs of this article were defrayed in partby page charge garian Academy of Sciences, H-1525 Budapest, Hungary. payment. This article must therefore be hereby marked "advertise- t Present address: Institute of Biochemistry and Biophysics, Polish ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Academy of Sciences, Warsaw 02532, Poland. 4884 Downloaded by guest on September 24, 2021 Biochemistry: Singer et aL Proc. Natl. Acad. Sci. USA 80 (1983) 4885 Table 1. Thermal denaturation of poly(dA-dT)-poly(dA-dT) containing 02-Me-dT or 04-Me-dT 15 mM NaCl/1.5 mM Na 0.1 M Tris-HCl Substitution Net citrate, pH 7 (pH 7.2) 1 M NaClt in poly(dA-dT) synthesis* t4,, 0C T,tC0 H,§ % tM, 0C T,t 0C t, 0(C T,t 0C H,§ % 9% 02-Me-dT 6-fold 61.0 2.2 74.7 5.2 22% 02-Me-dTt 7-fold 46.0 0.8 44.9 74.5 2.9 39.8 6% 04-Me-dTt 7-fold 46.0 0.8 45 74.7 2.6 40.0 13% 04-Me-dT 4-fold 61.3 1.8 75.3 3.0 None 46.2 1.0 41 62 2.0 74.8 3.1 39.9 Data are means oftwo to four determinations except where noted. Representative melting profiles are shown in Fig. 3A. All samples reannealed completely. * Uncorrected for losses in handling. tAlso contains 15 mM NaCl/1.5 mM Na citrate, pH 7. *Prepared by using the Klenow fragment of poll. §Temperature change between 25% and 75% of total hyperchromicity. INot mean of two to four determinations.

pyldeoxythymidine and benzene/isopropanol (9:1) for 04-iso- the same conditions as with pol I except 7 units of enzyme were propyldeoxythymidine. The molar extinction coefficients in water present in a 1-ml reaction mixture. The maximal hypochrom- are 02-isopropyldeoxythymidine (km. 229) E = 8.1 X 103, (Ama. icity was 35-40%. 258) E = 10.5 X 103; 04-isopropyldeoxythymidine (Ama. 281) 6 Isolation and Analysis of Polymers Synthesized with pol I. 6.8 X 103. Polymers in 0.1-0.2 ml of aqueous solution were separated from Two millimoles of 04-isopropyldeoxythymidine and 1.5 mmol salts and nucleotides by using a Bio-Gel P150 column (0.9 x 9 of 02-isopropyldeoxythymidine were converted to the methyl cm) with water as the eluant; 0.5-ml fractions were collected. derivatives by reaction in 5 ml of dry methanol with 3 mmol of The polymer eluted fairly sharply in fractions 5-8 while dNTPs sodium methoxylate for 48 hr at 37°C. After application to pre- trailed over fractions 10-25. For analysis, fractions 5-7 were parative silica gel plates, the methyldeoxythymidines were pu- pooled. rified by chromatographing three times with acetone/benzene Digestion to nucleosides was carried out as follows: To 0.1 (60:40). The Rf of 02-Me-dT (mp 152-154°C) was 0.07 and of A2w unit of polymer in 0.5 ml of 5 mM Tris-HCl (pH 7.5) con- 04-Me-dT (mp 174-1760C), 0.2. The yields were 36% and 52% taining 20 ,. of 0.1 M MgCl2 was added 5 Al of a mixture con- after recrystallization. The molar extinction coefficients in water are 02-Me-dT (Ama. 229) E = 7.1 X 103, (Ama, 257) E = 10.1 X 103; 04-Me-dT (Ama, 281) E = 6.7 X 103. A The triphosphates were prepared as the sodium salts by us- ing the general methods previously described for 0-alkyl-UDPs (14). The synthesis of the triphosphates followed the procedure of Hoard and Ott (15), using tributylammonium pyrophosphate as the phosphorylating agent. Limited digestion with bacterial alkaline phosphatase yielded the expected products: 0-meth- dT ylthymidine, 0-methyl-dTMP, and 0-methyl-dTDP. All spec- tra were identical to that of the parent methyl nucleoside (16). Synthesis of Alternating Poly(dA-dT, 02-Me-dT) and Poly(dA- dT, 04-Me-dT). Polymers were prepared by using either pol I or the Klenow fragment of pol I. A typical reaction mixture 0 using pol I contained (per ml) 50 ,.l of 1 M Tris HCI (pH 7.8), :0 20 ,ul of 0.1 M MgCl2, 0.15 A2w unit of poly(dA-dT), 0.1 ,umol of dATP, 0.1 /Lmol total of dTTP and o2_ or 04-Me-dTTP (1:1 or 1:3), and 1.5 ,ug of pol I (30 units). The reaction at 37C was followed by monitoring the decrease in absorbancy at 260 nm. The maximum hypochromicity was about 20% in 3 hr. The re- action was stopped by heating for 15 min at 75°C, and the dena- tured protein was removed by centrifugation (3,000 rpm, 30 K dAdo min). In some experiments, termed stepwise, the reaction mixture contained 0.1 ,umol of dATP, 25 nmol of dTTP, and 75 nmol of O-Me-dTTP. When the hypochromicity was maximal, an ad- ditional 25 nmol of dTTP was added, which then led to addi- 0 6 12 18 24 0 6 12 18 24 30 tional synthesis. This procedure was repeated until 0.1 tmol Time, min of dTTP had been added. Portions of the reaction mixture were taken at various times during this procedure to compare the FIG. 1. HPLC separation of nucleosides from enzyme digests of amount of O-Me-dT incorporated as a function of the extent of poly(dA-dT) and poly(dA-dT, 04-Me-dT). Detection of UV absorbancy reaction. was at 280 nm, the A.. of 04-Me-dT. The flow rate was 0.5 ml/min, These points were in the linear portion of the first 0.4 M ammonium formate (pH 4.7), 550C. (A) Deoxythymidine and step (5% hypochromicity), at the end of the first step (10% hy- deoxyadenosine (dAdo) from enzyme digest of poly(dA-dT). (B) Deoxy- pochromicity), and after the four steps (20% hypochromicity). thymidine, 04-Me-dT, and deoxyadenosine from enzyme digest of Polymers were prepared with the Klenow fragment by using poly(dA-dT, 04-Me-dT). This polymer contains 2% 04-Me-dT. Downloaded by guest on September 24, 2021 4886 Biochemistry: Singer et al. Proc. Nad Acad. Sci. USA 80 (1983)

A B C origin from dNTPs and small oligonucleotides. The polymer was then scraped off and eluted with water. Further purification dT could be achieved by using the column chromatography de- 02-Me-Thy scribed for poly(dA-dT). The yield of polymer was dependent on the primer and dNTP used for elongation. Alkylation of Poly(dA-dT)-Poly(dA-dT). Reaction with ['IC]- methylnitrosourea (NEN) and ['4C]ethylnitrosourea (Hoechst) dT was according to Jensen and Reed (19) and Jensen (20). Both [14C]alkylnitrosoureas were mixed with the unlabeled com- ¢ - dAdo pounds to give a specific activity of 0.25 mCi/mmol (1 Ci = 37 GBq) for the methyl compound and 1.7 mCi/mmol for the LO ~~~~~~~dAdo ethyl compound. After reaction was complete, the alkylated polymers were separated from reagent by column chromatog- raphy as described above. After three such separations, rerun- ning the peak tubes only, the specific activity was constant. To- tal methylation was 2.8% and ethylation was 3.5%. Thermal Denaturation Measurement. All measurements were made using Teflon-stoppered cuvettes in a dual-beam Varian Cary 219 spectrophotometer equipped with jacketed sample and reference chambers. The temperature of the cell compartment was regulated by a Neslab Eva-EndoCal pro- grammed at 10C/2 min. Up to four samples could be compared simultaneously. One-milliliter samples containing 0.1-0.2 A260 0 6 12 18 0 6 12 18 24 0 6 12 18 24 unit of polymer in water were dialyzed at 00C for 24 hr against Time, min the desired buffer. These included 15 mM NaCI/1.5 mM Na FIG. 2. Analysis of poly(dA-dT, 02-Me-dT) by HPLC after hydro- citrate, pH 7, 0.01 and 0.1 M Tris-HCI (pH 7.2), and 1 M NaCl lysis. Detection of UV absorbancy was at 254 nm. (A) 02-Me-dT was containing 15 mM NaCI/1.5 mM Na citrate, pH 7. heated for 5 hr at 950C, giving 75% depyrimidination to 02-methyl- thymine (02-Me-Thy). The nucleoside andbase are well separated. The shoulder eluting before 02-Me-dT is deoxythymidine, which results from RESULTS a small amount of dealkylation. (B) Deoxythymidine and deoxyaden- Incorporation of O2- and 04-Me-dTTP in Poly(dA-dT). 04_ osine from enzyme hydrolysis ofpoly(dA-dT). (C) Deoxythymidine, 02- and 02-Me-dTTP were utilized in with methylthymine, and deoxyadenosine from an enzyme digest of poly(dA- poorly synthesis poly(dA- dT, 02-Me-dT) after heating for 5 hr at 950C. Integration of the peaks dT) templates in the presence of dATP alone, since [3H]dATP gives the following composition: 50% deoxyadenosine, 43% deoxythy- was not detectably incorporated unless dTTP was present to- midine, 7% 02-Me-dT. When the results are corrected for incomplete gether with the modified dTTPs. On the other hand, with dTTP depyrimidination, the 02-Me-dT value is increased to 10% and the de- (25% of dATP) and O-Me-dTTP (75% of dATP), synthesis pro- oxythymidine value is decreased to 40%. ceeded the same extent as in the absence of O-Me-dTTP. When equal amounts of o2_ or 04-Me-dTTP and dTTP, total molarity taining 2.5 pug each-of DNase I, snake venom phosphodiester- equivalent to that of dATP, were used, standard conditions of ase, and bacterial alkaline phosphatase. Two hours at 3700 was synthesis with pol I resulted in 3- to 4-fold synthesis, which was sufficient for complete digestion as judged by the increase in similar to synthesis with equal molarity of dTTP and dATP. Un- absorbancy. der these conditions, 12-22% of the pyrimidines were 0-Me- HPLC analysis using a cation exchange column with elution dT. with 0.4 M ammonium formate (pH 4.7) was according to Kr6ger Variations were tried to determine whether it was possible and Singer (17). Typical HPLC profiles are shown in Figs. 1 and to either introduce more O-Me-dT or to increase the extent of 2. Because of the lack of sufficient separation of 02-Me-dT and synthesis. Stepwise addition of additional dTTP when synthesis deoxythymidine, the hydrolysates of 02-Me-dT-containing lagged or use of the Klenow fragment under competitive or polymers were heated in sealed tubes for 5 hr at 9500 to de- stepwise conditions increased the yield but not the proportion pyrimidate 02-Me-dT, which has a labile glycosyl bond (18). As of O-Me-dT. shown in Fig. 2A, 02-methylthymine is well separated from Since pol I can incorporate a single noncomplementary dNTP 02-Me-dT and deoxythymidine. Integration of the peaks in- terminally when there is no complementary dNTP (21), poly- dicated that 75% depyrimidination occurred. All values for o2_ mer was isolated for analysis both during the linear portions of Me-dT include this correction. The composition of polymers the synthesis and after synthesis had plateaued. With pol I and containing O-Me-dT shows that, within experimental error, de- 04-Me-dTTP (dTTP/04-Me-dTTP/dATP = 25:75:100), there oxythymidine is decreased by the amount of O-Me-dT. was 8% incorporation with 0.6-fold increase in polymer and 10.8% Synthesis by Terminal Deoxynucleotidyltransferase of Sin- incorporation with 1.5-fold synthesis. When the Klenow frag- gle-Stranded Deoxypolynucleotides Containing O-Me-dT. A ment and 02-Me-dTTP (in the same proportion) were used, 0.5-ml reaction mixture contained 0.2 ml of 0.5 M cacodylate identical values of 6.4% 02-Me-dT were obtained with 2-fold buffer (pH 7.2), 40 p1 of 0.1 M MgCl2, 6 ,u of 0.1 M 2-mer- and 7-fold synthesis. It is thus unlikely that O-alkyl-dT is in- captoethanol, 0.05 A260 unit of oligo(dA) or (dC) or (dT)12 18, 0.8 corporated only when there is no dTTP left. ,tmol of dNTP, 0.2 ,mol of o2_ or 04-Me-dTTP, and 750 units Synthesis with Terminal Deoxynucleotidyltransferase of o2- of TdT. After incubation at 37°C for 24 hr, the mixture was and04-Methylthymidine-ContainingPolymers. Oligo(dT)12-18, evaporated to 50 ,ul, spotted on cellulose thin-layer sheets oligo(dC)12-18, and oligo(dT)12 18 were all used as primers and (Eastman No. 6065), and developed in n-propanol/NH40H/ elongated with dTTP or dCTP or with dATP and either 04-Me- water (55:10:35). The TLC were dried and again developed to dTTP or 02-Me-dTTP, generally in a unmodified/modified ra- completely separate the high molecular weight polymer at the tio of 5:1. Larger amounts of O-Me-dTTP inhibited reaction. Downloaded by guest on September 24, 2021 Biochemistry: Singer et al. Proc. Natl. Acad. Sci. USA 80 (1983) 4887 Table 3. Fidelity of DNA synthesis with poly(dA-dT) containing 02 or 04-Me-dT dGMP/dAMP X 104* Average Polymer 1 2 error rate (dA-dT) 0.49 0.33 1/50,100 (dA-dT, O4-Me-dT)t 1.32 1.8 1/13,500 (dA-dT) 0.35 0.68 1/42,700 A (dA-dT, 02-Me-dT)t 0.59 1.52 1/26,300 b The reaction mixture (10-50 1.l) contained, per ml: 50 AuM Tris HCl (pH 7.8), 2 juM MgCl2, 60 ,uM dTTP, 0.OO5-0.015A2po unit ofpolymer, a 20 units ofpol I, and either 60 AuM dATPand 6 ,uM [3H]dGTP (16.2 Ci/ mM) or 60 pM [3HldATP (120 mCi/mM) and 6 kLM dGTP. Incubation o was for 60 min at 370C. [3H]dATP incorporation was 20-40 nnol/ml. c9 Poly(dA-dT 04-Me-dT) * Each entry in experiments 1 and 2 represents the average of six val- ues. 04-Me-dT. Poly(dA-dT, t2.8% 02_Me-dT) t6.0% 02-Me-dT. Poly(dA-dT) with the same melting temperature (tm) as poly(dA-dT) (5). This 45 is attributed in part to the destabilization resulting from the 40 45 50 40 energy necessary to maintain the methyl in the trans or anti Temperature, 0C conformation (5). On this basis, it can be assumed that the methyl on the of must be oriented anti. The methyl FIG. 3. Thermal melting profiles in 15 mM NaCl/1.5 mM Na ci- group 04 thymidine trate, pH 7. (A) Polymers synthesized by using the Klenow fragment group on the o2 of thymidine could be sterically hindering if of DNA polymerase I. (See also Table 3.) Curves: a, poly(dA-dT), tm = it were facing into the hydrogen bonding side. Since the tm is 46.10C; b, poly(dA-dT, 02-Me-dT), tm = 46.00C, 18% of the pyrimidines unchanged, this substituent is probably also anti. are 02-Me-dT; c, poly(dA-dT, 04-Me-dT), tm = 46.00C, 5.6% of the py- Destabilization of poly(dA-dT) also occurs measurable when rimidines are 04-Me-dT. (B) Various alkylated poly(dA-dT)s. Curves: the polymer is methylated or ethylated (Table 2 and Fig. 3B). a, the polymer was 3.5% ethylated by treatment with ethylnitrosourea, In these cases, neither N6-alkyl- nor 0-alkylthymidines are t4m = 45.8°C; b, the polymer was 2.5% methylated by treatment with methylnitrosourea, t,, = 45.5°C; c, unmodified poly(dA-dT), t4, = 46.3°C. formed to more than 0.1% of the bases. Mideoding Studies of Deoxypolymers Containing O-Me-dT. The possibility that 0-Me-dT led to misincorporation was stud- dATP was by far the best incorporated normal triphosphate, ied by copying the synthesized polymers with pol I (9) or tran- regardless of the primer used, while dTTP was the poorest. scribing them with E. coli DNA-dependent RNA polymerase With 20% input of 0-Me-dTTP, the polymers synthesized con- (1). Data for misincorporation of G by pol I are shown in Table tained 5-8% 0-methylthymidine, indicating that they were 3. The presence of 2.8% 04-Me-dT led to an approximately 3- substrates for terminal deoxynucleotidyltransferase. The great- to 4-fold increase in dGMP as compared with poly(dA-dT), while est elongation of the primer, as judged by optical density, was with 6.9% 0-Me-dT the increase in misincorporation was lower 10-fold, which would mean elongation by 120-180 nucleotides. (=2-fold). Single-stranded polymers prepared by terminal de- The relatively high molecular weight was also shown by a 60- oxynucleotidyltransferase misincorporated about 1 0-Me- 70% increase in absorbancy on enzyme digestion to nucleo- G/15 sides. dT when transcribed (data not shown). Thermal Stability of Poly(dA-dT, 04-Me-dT), Poly(dA-dT, 02-Me-dT), and Alkylated Poly(dA-dT). Melting curves were DISCUSSION obtained with polymers containing 9-22% of the thymidine An important test of whether a modified base has changed residues displaced by O-Me-dT (Table 1 and Fig. 3A). All of properties is whether, as the triphosphate, it can substitute for the parameters, in three different buffers, were, within ex- a normal base in enzymatic synthesis and, if incorporated, perimental error, the same as those of poly(dA-dT) (22). This whether its presence would lead to transitions or transversions. indicates that there is no detectable structural alteration re- In fact, synthesis of either a random polymer (with terminal sulting from a considerable number of modified thymidine res- deoxynucleotidyltransferase) or a defined polymer (with DNA idues. The increased width of the thermal transition, observed polymerases) containing simple alkyl derivatives is highly de- with all polymers, is considered to be a proof of the alternating pendent on the specific derivative and the enzyme. structure (23). In contrast, similar polymers containing 13-18% Terminal deoxynucleotidyltransferase-directed polymeriza- N6-methyldeoxyadenine do not melt with this cooperativity or tion of dNTPs does not mimic cellular replication, although it

Table 2. Thermal denaturation of alkylated poly(dA-dT) poly(dA-dT) 15 mM NaCl/1.5 mM Na 0.1 M Tris HCl Alkylated citrate, pH 7 0.01 M Tris HCl (pH 7.2) (pH 7.2) 1 M NaCl* poly(dA-dT) tm, °C T. 0C H,t % tm °C T, 0C H,t % tm, 0C T, 0C tin °C T, 0C 2.8% methylated 45.5 2.0 34.3 50.3t 2.0 34.5 60.7 2.6 73.8 5.2 3.5% ethylated 45.7 1.9 30.8 50.8t 1.9 32.3 No treatment 46.2 1.0 41 51.7t 1.0 39.9 62.0 2.0 75 3.1 Data are means oftwo to four determinations exceptwhere noted. Representative meltingprofiles are shown in Fig. 3B. Poly(dA-dT) was alkylated with methylnitrosourea or ethylnitrosourea. For footnotes, see Table 1. Downloaded by guest on September 24, 2021 4888 Biochemistry: Singer et aL Proc. Natl. Acad. Sci. USA 80 (1983) is not completely unspecific. This is also true to a lesser extent the total alkylation, may account for destabilization. for synthesis of a defined polymer by error-prone polymerases, The present results cannot be regarded as directly relevant such as that from avian myeloblastosis virus and from eukary- to in vitro systems (27, 28) or those using extracts containing a otes that lack the proofreading function. In contrast, pol I ex- replicating system (29). However, the finding that the 0-Me- hibits high fidelity by excising noncomplementary nucleotides dTTPs can substitute for dTTP, using an enzyme capable of ex- (4). Therefore, the fiact that 06-methyl dGTP and 06-ethyl dGTP cising mismatched nucleotides, suggests that in vivo possible could be incorporated into polymers by terminal deoxynucleo- alkylated deoxythymidines or dTTPs may be incorporated into tidyltransferase but not by pol I (8, 10) indicates that either no DNA without altering the structure. This may account for the incorporation occurred or that once incorporated the nucleotide low rate of repair (12, 30), and it may increase the probability was excised. Abbott et aL (10) have evidence that 06-methyl of misincorporation during either transcription or replication. dGTP is not incorporated by pol I. Copolymers containing 06- methyl or 06-ethyldeoxyguanosine have been prepared but only This work was supported by Grant CA 12316 from the National Can- with terminal deoxynucleotidyltransferase, although both de- cer Institute. rivatives inhibit polymerization of unmodified dNTPs (7, 8, 10). 1. Kulmierek, J. T. & Singer, B. (1982) Biochemistry 21, 5723-5728. There seem to exist no data on the successful incorporation 2. Singer, B. (1982) in Molecular and Cellular Mechanisms of Mu- tagenesis, eds. Lemontt, J. F. & Generoso, W. M. (Plenum, New into an alternating copolymer of a N- or 0-alkylated dNTP, with York), pp. 1-42. the exception of N6-methyl-dATP (5). N4-Hydroxy-dCTP has 3. Gerchman, L. L. & Ludlum, D. B. (1973) Biochim. Biophys. Acta been shown to be a substrate for pol I in site-directed muta- 308, 310-316. genesis (24) but it, like N6-methyl-dATP, can form normal hy- 4. Loeb, L. A. & Kunkel, T. A. (1982) Annu. Rev. Biochem. 51, 429- drogen bonds (25). Similarly, as expected, 5-ethyl-dUTP can 457. substitute for dTTP (6). In the case of the polymers we 5. McGhee, J. D. & von Hippel, P. H. (1977)J. Biol, Chem. 16, 3276- have 3293. made, the hydrogen on the N-3 is lost and thus that hydrogen 6. Sigi, J., Szabolcs, A., Szemzo, A. & Otvos, L. (1977) Nucleic Acids bonding ability is also lost, so that only a single bond can be Res. 4, 2767-2777. formed with adenosine. Nevertheless, the tm value, width of 7. Mehta, J. R. & Ludlum, D. B. (1978) Biochim. Biophys. Acta 521, transition, and hyperchromicity are indistinguishable from those 770-778. of poly(dA-dT). These data indicate that the polymers synthe- 8. Muller, R., Drosdziok, W. & Rajewsky, M. J. (1981) Carcinogen- sized with 6-22% of the deoxythymidine replaced by O2_ or 04_ esis 2, 321-327. 9. Singer, B., Kusmierek, J. T. & Fraenkel-Conrat, H. (1983) Proc. Me-dT are high molecular weight duplexes. Thus, single hy- NatL Acad. Sci. USA 80, 969-972. drogen bonds occurring to this extent in a well-stacked polymer 10. Abbott, P. J., Mehta, J. R. & Ludlum, D. B. (1980) Biochemistry of relatively high stability do not destabilize the helix. Possible 19, 643-647. looping out resulting from the modified base can be excluded 11. Singer, B. (1979) J. Nati Cancer Inst. 62, 1329-1339. by the cooperativity of the tm and the high hyperchromicity (25, 12. Singer, B., Spengler, S. & Bodell, W. J. (1981) Carcinogenesis 2, 26). Clustering of 0-methylthymidine is unlikely because the 1069-1073. 13. Scherer, E., Timmer, A. P. & Emmelot, P. (1980) Cancer Lett. early linear stage of synthesis yielded polymers of approxi- 10, 1-6. mately the same composition as found at the termination of re- 14. Singer, B., Fraenkel-Conrat, H. & Kuimierek, J. T. (1978) Proc. action. In addition, all of the polymers are fully rehybridized Natl Acad. Sci. USA 75, 1722-1726. after thermal denaturation, indicating a completely reversible 15. Hoard, D. E. & Ott, G. (1965) J. Am. Chem. Soc. 87, 1785-1788. structure. Interestingly, poly[dA, N6-Me-dA-dT] has a much 16. Kusmierek, J. T. & Singer, B. (1976) Nucleic Acids Res. 3, 989- lower tm with less cooperativity than poly(dA-dT), even 1000. though 17. Krbger, M. & Singer, B. (1979) Biochemistry 18, 3493-3500. the N6-methyladenosine residues are apparently hydrogen 18. Singer, B., Kroger, M. & Carrano, M. (1978) Biochemistry 17, bonded normally to thymidine (5). This is attributed to disrup- 1246-1250. tion of stacking by the methyl group. 19. Jensen, D. E. & Reed, D. J. (1978) Biochemistry 17, 5098-5107. In contrast to incorporation of a specific modified deoxy- 20. Jensen, D. E. (1978) Biochemistry 17, 5108-5113. thymidine, poly(dA-T) methylated or ethylated to a much lower 21. Agarwal, S. S., Dube, D. K. & Loeb, L. A. (1979)J. Biol Chem. 254, 101-106. extent was slightly less stable under all conditions studied. Both 22. Szybalski, E. H. & Szybelski, W. (1975) in Handbook ofBiochem- the cooperativity and the hyperchromicity were also affected. istry and Molecular Biology. Nucleic Acids, ed. Fasman, G. D. The nitrosoureas used for modification of poly(dA-dT) primar- (CRC, Boca Raton, FL), Vol. 1, pp. 575-588. ily alkylate the phosphodiesters. o2- and 04-Me-dT represent 23. Wells, R. D., Larson, J. E., Grant, R. C., Shortle, B. E. & Can- only 4% of the total methylation, with 30% and 3%, respec- tor, C. R. (1970)J. Mold Biol 54, 465-497. tively, of o2- and 04-Me-dT (19, 20). In ethylnitrosourea-treated 24. Mfiller, W, Weber, H., Meyer, F. & Weissmann, C. (1978)1 Mol, Biol 124, 343-358. poly(dA-T), 2% of the ethyl groups are 1-ethyldeoxyadenine 25. Spengler, S. & Singer, B. (1981) Biochemistry 20, 7290-7294. and 30% are 02-ethyldeoxythymidine (19). In the case of meth- 26. Nelson, J. W., Martin, F. H. & Tinoco, I., Jr. (1981) Biopolymers ylnitrosourea-treated poly(dA-dT), only 0.1% of the thymidine 20, 2509-2531. residues were 0-methyl (20). Therefore other modifications than 27. Saffhill, R. & Fox, M. (1980) Carcinogenesis 1, 487-493. those of the oxygen of thymidine must be responsible for the 28. Brennand, J., Saffhill, R. & Fox, M. (1982) Carcinogenesis 3, 219- observed change in stability. Although it can be argued that 222. 29. Dodson, L. A., Foote, R. S., Mitra, S. & Masker, W. E. (1982) methylation of the N-3 of deoxythymidine or N-1 of deoxy- Proc. Natl Acad. Sci. USA 79, 7440-7444. adenosine would eliminate any hydrogen bond, these modifi- 30. Swenberg, J. A., Bedell, M. A., Billings, K. C., Huh, N., Kir- cations are not detected in methylated poly(dA-dT). Thus it is stein, U. & Rajewsky, M. F. (1983) Proc. Am. Assoc. Cancer Res. more likely that alkylphosphotriesters, which are 60-75% of 24, 68. Downloaded by guest on September 24, 2021