Protonated base pairs explain the ambiguous pairing properties of 06 - methylguanine

The base-pairing interactions of promutagenic 06-methylguanine (06-MeGua) with cytosine and thymine in deuterated chloroform were investigated by 1H NMR spectroscopy. Nucleosides were derivatized at hydroxyl positions with triisopropylsilyl groups to obtain solubility in nonaqueous solvents and to prevent the ribose hydroxyls from forming hydrogen bonds. We were able to observe hydrogen-bonding interactions between nucleic acid bases in a solvent of low dielectric constant, a condition that approximates the hydrophobic interior of the DNA helix. O6-MeGua was observed to form a hydrogen-bonded mispair with thymine. Whereas 06-MeGua did not form hydrogen bonds with cytosine (via usual, wobble, or unusual tautomeric structures), it did form a 1:1 hydrogen-bonded complex with protonated cytosine. The pairing of unprotonated cytosine in chloroform is thus consistent with the known preference of 06-MeGua for thymine over cytosine in polymerase reactions. In contrast, the pairing of protonated cytosine is consistent with the greater stability of oligonucleotide duplexes containing cytosine O6-MeGua as compared with thymineO6-MeGua base pairs [Gaffney, B. L., Markey, L. A. & Jones, R. A. (1984) Biochemistry 23, 56865691]. Our observation that cytosine must be protonated in order to pair with O6-MeGua suggests that the cytosine 06MeGua base pair in DNA is stabilized by protonation of cytosine. Through this mechanism, methylation at the 06 position of guanine in double-stranded DNA could promote cross-strand deamination of cytosine (or 5-methylcytosine) to produce uracil (or thymine). As first observed by Loveless (1), 06 guanine adducts in DNA are particularly important in induction of carcinogenesis (reviewed in ref. 2). The mutagenic potential of guanine alkylation at the 06 position appears to be determined both by its effects on the hydrogen-bonding properties of guanine and by the persistence of the lesion (3). In vitro studies have shown that 06-methylguanine (06-MeGua) codes for thymine (or uracil) instead of cytosine during both DNA replication and transcription processes (4-8). In DNA synthesis, dTTP is incorporated at least an order of magnitude more frequently than dCTP opposite 06-MeGua (4). Conversely, 06-MedGTP is incorporated preferentially opposite thymine rather than cytosine by the Klenow fragment of Escherichia coli DNA polymerase I (5, 6). In transcription, UTP is incorporated preferentially over CTP opposite 06-MeGua by RNA polymerase (7, 8). The relative contributions of the four O6-MeGua base pairs (with guanine, adenine, cytosine, or thymine) to oligonucleotide stability are in contradiction with the coding preferences of 06-MeGua. Jones and coworkers (9) have reported that in oligodeoxynucleotide dodecamers the 06-MeGua thymine base pair has a greater destabilizing effect on duplex stability than does any other 06-MeGua base pair, whereas the 06-MeGuacytosine base pair is the least destabilizing. Thus, although 06-MeGua interacts preferentially with thymine in the polymerase complex, it forms the least stable base pair with thymine in oligonucleotides. Such results imply either that base pairing is not as important as generally assumed in replication or that factors which stabilize 06_ MeGua base pairing in the polymerase complex differ significantly from those in the DNA helix. In two recent reports, Patel et al. (10, 11) deduced by 1H NMR that both the O6-MeGua-thymine and the 06-MeGua'cytosine base pairs are stacked in dodecamers. These authors were not able to establish direct evidence regarding the specific hydrogenbonding schemes of 06-MeGua with either cytosine or thymine. To understand the pairing properties of 06-MeGua in the absence of stacking effects and to reconcile the discrepant effects of 06 methylation of guanine on the structure and biosynthesis of DNA, we have investigated the base-pairing interaction of 06-MeGua with thymine and cytosine in C2HC13 with 1H NMR. Numerous spectroscopic and crystallographic studies have shown that the hydrogen-bonding specificity expressed by nucleic acid bases in DNA is manifested between nucleic acid monomers in nonaqueous solvents (12-16). To date, however, the poor solubilities of nucleosides and bases in appropriate solvents have impeded physicochemical investigations of the base-pairing properties of the bases in solvents where solutesolute hydrogen bonding can be maximized. Here we report that the 2',3'-isopropylidine-5'-triisopropylsilyl derivatives of cytidine (compound 1) and 06-methylguanosine (compound 2) and the 3',5'-bis(triisopropylsilyl) derivative of thymidine (compound 3) shown in Fig. 1 have properties that are suitable for spectroscopic investigation of the basepairing properties of nucleic acid bases in solution. These nucleoside derivatives are soluble in chloroform at concentrations on the order of 50 mM at temperatures approaching -40° C, thus allowing direct evaluation of the effects of methylation on DNA base pairing, as described in the experiments reported below. The purpose of the triisopropylsilyl and isopropylidine groups is to obtain solubility ofthe nucleosides and to prevent the ribose hydroxyls from forming hydrogen bonds. MATERIALS AND METHODS Synthesis. Guanosine and cytidine (American Bionetics, Emeryville, CA) were converted in 10to 20-g lots to the 2' ,3 '-isopropylidine nucleosides (17). The 2' ,3 '-isopropylidine nucleosides were added to a 1.2-fold molar excess of triisopropylsilyl chloride (Aldrich) in the presence of excess imidazole in dimethylformamide to form the 2',3'isopropylidine-5'-triisopropylsilyl nucleosides (18). Thymidine was converted to 3',5'-bis(triisopropylsilyl)thymidine Abbreviation: 06-MeGua, 06-methylguanine. *To whom reprint requests should be addressed. 1779 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 1780 Biochemistry: Williams and Shaw (Fisher) in the dark under argon. Flash chromatography was performed with 230to 400-mesh silica gel (Merck). 1H NMR spectra were obtained on Brucker WM 250 and Varian XL-300 NMR spectrometers. Typically, 48 transients were accumulated over 4000-5000 Hz, using 16K, double-precision (32-bit) data points. Chemical shifts are in reference to the CHCl3 (7.243 ppm) impurity contained in the deuterated chloroform. The probe temperatures were calibrated with the chemical shifts of methanol (20).