5-Hydroxymethyl-2'-deoxyuridine ( ^hm dU) was prepared by the method of Shiau et al . ( 15 ). 2-Cyanoethyl alcohol, trifluoroacetic acid, acetic anhydride, pyridine, 1,2,4-triazole, triethylamine, 1,4-dioxane, trimethysilyl chloride, benzoyl chloride, sodium sulfate, DMAP (4-dimethylaminopyridine), 4,4'-dimethoxytrityl chloride, 2-cyanoethyl- N , N -diisopropylchlorophosphoramidite, methanol, dichloromethane, toluene and silica gel were obtained from Aldrich. Silica gel H was obtained from Fluka and DNA grade G25 resin was obtained from Pharmacia. Ammonium hydroxide (29.8%) was obtained from Mallinckrodt. Normal deoxynucleoside phosphoramidites and solid supports were obtained from Glen Research. Nuclease P1 and bacterial alkaline phosphatase were obtained from Sigma. Restriction endonucleases Msp I and Hpa II and appropriate enzyme buffers were obtained from New England BioLabs. T4 Polynucleotide kinase was obtained from the United States Biochemical Corporation and ³² P-ATP was obtained from ICN.

Silica gel chromatography was performed with methanol in dichloromethane (1-10%) and thin layer chromatography was performed with glass-backed silica plates embedded with fluorescent indicator. Elemental analysis was performed by Desert Analytics, Tucson, Az, USA.

HPLC was performed with a Perkin-Elmer series 4 liquid chromatograph interfaced with an LKB 2140 spectral diode array detector. UV spectra were recorded with a Perkin-Elmer lambda 3B UV/Vis spectrophotometer. GC/MS analysis was performed with a Hewlett-Packard 5890 gas chromatograph interfaced with a 5970 series mass selective detector. Nuclear magnetic resonance (NMR) spectra were recorded with a Varian Unity 300 spectrometer. DNA synthesis was performed with a Pharmacia Gene Assembler. Gels from restriction nuclease digests were visualized with a Molecular Dynamics PhosphorImager.

Nucleosides synthesis

5-(2-cyanoethoxy)methyl-2 ' -deoxyuridine ( 2 ). 5-Hydroxymethyl- 2'-deoxyuridine (500 mg, 1.93 mmol) in 2-cyanoethyl alcohol (10 ml) was treated with a catalytic amount of trifluoroacetic acid (100 [mu]l). The solution was stirred at 130^oC under vacuum for 1 h. The product was then isolated by silica gel chromatography. Obtained, 420 mg (70% yield); 1H NMR (300 MHz, d6 DMSO) [delta], 11.42 (s, NH), 7.94 (s, H6), 6.16 (t, H1', J=6.0 Hz), 5.26 (d, 3'OH), 5.04 (t, 5'OH), 4.24 (m, H3'), 4.17 (s, 5-CH ₂ O-), 3.78 (m, H4'), 3.58 (m, H5', H5'',C H ₂ CH ₂ CN), 2.75 (t, CH ₂ C H ₂ CN, J=6.3 Hz), 2.12 (m, H2',H2''). (Found: C, 50.06; H, 5.66; N, 13.35; O, 31.27; Calc. for C ₁₃ H ₁₇ N ₃ O ₆ : C, 50.16; H, 5.51; N, 13.49: O, 30.84%) 5-(2-cyanoethoxy)methyl-2 ' -deoxycytidine ( 3 ). 5-(2-Cyanoethoxy)methyl-2'-deoxyuridine ( 2 ) was converted to 5-(2-cyanoethoxy)methyl-2'-deoxycytidine ( 3 ) by the method of Divakar and Reese ( 16 ). 5-(2-Cyanoethoxy)methyl-2'-deoxyuridine (400 mg, 1.3 mmol) was dried by coevaporation of dry pyridine. Pyridine (50 ml) was then added, followed by dropwise addition of acetic anhydride (1.23 ml, 13 mmol). The solution was stirred at room temperature for 3 h. Anhydrous methanol (5 ml) was added and the mixture was stirred for an additional 5 min. Solvents were removed under reduced pressure and the product was dried to a foam by coevaporation of toluene. The acetylated derivative was used directly for the next conversion.

A separate mixture containing 1,2,4-triazole ( 0.81 g, 11.7 mmol) and acetonitrile (20 ml) was stirred at 0^oC, and treated by dropwise addition of phosphorus oxychloride (242 [mu]l, 2.6 mmol), followed by triethylamine (1.6 ml, 11.2 mmol). The dry acetylated nucleoside was dissolved in a minimal volume of acetonitrile and added to the triazole mixture. Stirring was continued for 1 h. Solvents were removed under reduced pressure and the residue was dissolved in dichloromethane and washed with brine and distilled water. The organic phase was dried with sodium sulfate and filtered. Solvents were removed under reduced pressure and the product was dissolved in 1,4-dioxane (10 ml) and treated with 29% aqueous ammonia (2 ml). The mixture was stirred at room temperature for 2 h. Solvents were removed under reduced pressure and the product was isolated by silica gel chromatography. Obtained, 305 mg (76% yield); 1H NMR (300 MHz, d6 DMSO) [delta], 7.85 (s, H6), 7.44 (s, NH), 6.68 (s, NH) 6.15 (t, H1', J=6.0 Hz), 5.22 (d, 3'OH), 5.02 (t, 5'OH), 4.23 (m, H3', 5-CH ₂ O-), 3.78 (m, H4'), 3.58 (m, H5', H5'', -CH ₂ C H ₂ CN), 2.78 (m, -C H ₂ CH ₂ CN), 2.03 (m, H2', H2''). N ⁴ -benzoyl-5-(2-cyanoethoxy)-methyl-5 ' -O-(4-4 ' -dimethoxytrityl)-2 ' -deoxycytidine-3 ' -O-(2-cyanoethyl)- N , N -diisopropylphosphoramidite ( 4 ). 5-(2-Cyanoethoxy)methyl-2'-deoxycytidine ( 3 ) was converted to N ⁴ -benzoyl-5-(2-cyanoethoxy)methyl-2'-deoxycytidine by the method of Ti et al . ( 17 ). 5-(2-cyanoethoxy) methyl-2'-deoxycytidine (1 g, 3.23 mmol) was dried by coevaporation of dry pyridine. Pyridine (16 ml) was added, followed by trimethysilyl chloride (2.06 ml, 16.2 mmol). After 15 min at room temperature, the stirred solution was treated with benzoyl chloride (1.9 ml, 16.5 mmol). The solution was continuously stirred for an additional 3 h. The mixture was then cooled to 0^oC and water (3 ml) was added. After 5 min, 29% aqueous ammonia (4 ml) was added and the mixture was stirred at room temperature for 15 min. Solvents were removed under reduced pressure and the residue was dissolved in dichloromethane and washed with saturated aqueous sodium bicarbonate and brine. The organic phase was dried with sodium sulfate and filtered. Solvents were removed under reduced pressure and the product was isolated by silica gel chromatography. Obtained, 0.95 g (71% yield); 1H NMR (300 MHz, d6 DMSO) [delta], 8.20-8.25, 7.52-7.65 (m, aromatic), 7.90 (s, H6), 6.18 (t, H1', J=6.0 Hz), 5.32 (d, 3'OH), 5.12 (t, 5'OH), 4.47 (m, H3'), 4.29 (m, 5-CH ₂ O-), 3.87 (m, H4'), 3.55-3.70 (m, H5', H5'', C H ₂ CH ₂ CN), 2.79 (t, CH ₂ C H ₂ CN), 2.25 (m, H2', H2'').

N ⁴ -benzoyl-5-(2-cyanoethoxy)methyl-2'-deoxycytidine was converted to the protected 5'-dimethyoxytrityl-3'-phosphoramidite by standard methods ( 18 ). Dimethoxytrityl was added to the 5' hydroxyl group using 4, 4'-dimethoxytrityl chloride and DMAP in pyridine. The product was washed with brine, extracted with dichloromethane and purified by silica gel chromatography. The purified trityl derivative was converted to the 3'-phosphoramidite using 2-cyanoethyl- N , N -diisopropylchlorophosphoramidite and diisopropylethylamine in dry acetonitrile and isolated using silica gel H. 5-(Hydroxymethyl)-2 ' -deoxycytidine ( 5 ). In order to generate an authentic marker of ^hm dC, 5-(2-cyanoethoxy)methyl-2'-deoxycytidine ( 3 ) was treated with aqueous ammonia at 60^oC overnight. Quantitative conversion to ^hm dC was observed by TLC. 1H NMR (300 MHz, d6 DMSO) [delta], 7.75 (s, H6), 7.34 (s, NH), 6.59 (s, NH) 6.17 (t, H1', J=6.0 Hz), 5.20 (d, 3'OH), 5.00 (m, 5'OH, CH ₂ O H ), 4.18 (m, H3', 5-C H ₂ OH), 3.77 (m, H4') 3.56 (m, H5', H5''), 1.99 (m, H2', H2'').

Oligonucleotide synthesis and purification

The ^hm C-phosphoramidite ( 4 ) was dissolved in dry acetonitrile and placed in one of the additional ports of the DNA synthesizer. The standard 1.3 [mu]mol synthesis cycle with retention of the 5' terminal dimethoxytrityl group was used without modification. Following synthesis, the solid support was placed in concentrated aqueous ammonia in a sealed vial and heated at 65^oC for 60 h. The trityl-containing oligonucleotide was purified by HPLC on a Hamilton PRP semipreparative column using 0.1 M triethyl ammonium acetate and an acetonitrile gradient. The purified trityl containing oligonucleotide was detritylated in 80% acetic acid at room temperature for 30 min. The aqueous acetic acid was removed under reduced pressure. The residue was dissolved in water, the pH was adjusted to 7 and extracted with ethyl acetate. The aqueous phase containing the detritylated oligo was concentrated under reduced pressure and repurified by reverse phase HPLC.

Sequences of oligonucleotides synthesized were as follows.

Test sequence: 5'(TG ^hm CATG ^hm CATG ^hm CA);

Upper strand: 5'(TACCCAGGAATTC C/C GGGATATCCTGG);

Lower strand: 5'(CCAGGATATC C/C GGGAATTCCTGG).

Single cytosine residues at the Msp I/ Hpa II cut site (bold) were replaced with either 5 ^m C or ^hm C in the upper strand and in the lower strand with 5 ^m C (Table 1 ).

Table 1 . Recognition sites of the duplex oligonucleotides probed with the Msp I/ Hpa II restriction endonucleases The complete sequences of the upper and lower strands are given in Materials and Methods.

Analysis of base composition

The purified oligonucleotides were enzymatically digested using nuclease P1 and bacterial alkaline phosphatase ( 19 ). The liberated deoxynucleosides were separated by HPLC using a reverse phase column and a gradient of 0.05 M sodium phosphate, pH 4.0 with increasing methanol (0 to 20%). Detection was provided by a photodiode array detector. The deoxynucleosides ^hm dC, dT, dG, and dA are chromatographically separable and identifiable based upon their characteristic UV spectra. Retention times, molar absorbtivity (pH 4.0, 265 nm, M ^-1 cm ^-1 ) and absorbance maxima (nm) for the deoxynucleosides are ^hm dC, 2.8 min, 11.0 * 10 ³ , 274 nm; dG, 7.6 min, 12.3 * 10 ³ , 252 nm; dT, 8.7 min, 9.5 * 10 ³ , 265 nm; dA, 11.8 min, 13.0 x 10 ³ , 259 nm. All deoxynucleosides were present in the correct ratios based upon integration of the HPLC chromatogram at 265 nm.

The oligo was also acid hydrolyzed to liberate the constituent free bases. The bases were silylated and analyzed by GC/MS as previously described ( 20 ). Bases were identified by their characteristic retention times and mass spectra. Observed retention times (min) and most abundant ion ( m/z ) for the silylated derivatives are thymine, 9.4 min, m/z 255; cytosine, 10.9 min, m/z 254; 5 ^m C 11.3 min, m/z 254; ^hm C 13.7 min, m/z 357; adenine 14.9 min, m/z 264; and guanine, 17.6 min, m/z 352. Solutions of known concentration of the free bases were used to generate standard curves relating base concentration to integrated chromatogram peak areas at appropriate mass ion currents. Based upon these standard curves, we were able to verify that all bases were present in synthetic oligonucleotides in correct ratios.

Restriction enzyme cleavage of the oligonucleotide

Portions of the oligonucleotides were ³² P-end labelled and purified by G25-spin columns. Duplexes were prepared by combining equimolar portions of the upper and lower oligonucleotide strands in restriction nuclease buffer, heating for 5 min at 90^oC followed by slow cooling. Restriction enzyme (20 U) was added and cleavage reactions were allowed to proceed for 15 to 30 min at 37^oC. Oligonucleotide duplexes were precipitated with ethanol, resuspended in the gel loading buffer and analyzed by gel electrophoresis on a 20% polyacrylamide denaturing gel. Gels containing labelled oligonucleotides were visualized with a PhosphorImager.

RESULTS

Previously, we presented a strategy for the selective protection of the 5-hydroxymethyl group of ^hm dU and placement of ^hm U residues in synthetic oligonucleotides ( 21 ). It was envisaged that ^hm C residues could be incorporated conveniently by formation of the 4-triazole derivative of the ^hm U-phosphoramidite as described for other 4-substituted pyrimidines ( 22 ). In our hands, however, conversion of the ^hm U-phosphoramidite to the corresponding triazole derivative did not go to completion, perhaps due to a steric problem introduced by the acetylated hydroxymethyl group in the adjacent 5-position. Attempts to separate the triazole phosphoramidite by silica gel chromatography resulted in substantial generation of starting material. As a second strategy, we attempted to selectively acetylate the 5-hydroxymethyl group of 5-hydroxymethyl-2'-deoxycytidine ( ^hm dC) as described previously for ^hm dU ( 21 ); however, we observed quantitative cleavage of the glycosidic bond.

As an alternative strategy, we exploited the known reactivity of the 5-hydroxymethyl group of ^hm dU to condense with alcohols as well as carboxylic acids ( 21 , 23 , 24 ). ^hm dU was reacted with cyanoethyl alcohol in the presence of a catalytic amount of trifluoroacetic acid to form the 5-cyanoethyl ether in high yield. The cyanoethyl derivative of ^hm dU quantitatively regenerates ^hm dU by [beta]-elimination when treated overnight with aqueous ammonia at 60^oC. The cyanoethyl group is routinely used as a phosphate protecting group for oligonucleotide synthesis ( 18 ) and Christopherson and Broom ( 25 ) have used the cyanoethyl group for base protection in the synthesis of 2'-deoxy-6-thioguanosine-containing oligonucleotides.

The 5-protected ^hm dU derivative (Fig. 2 , 2 ) formed by reaction of ^hm dU with cyanoethanol was then converted to the corresponding deoxycytidine derivative by the method reported by Divakar and Reese ( 16 ). Fortunately, the 4-triazole group is displaced by ammonia at room temperature whereas the cyanoethyl protecting group remains intact. The 5-cyanoethyl protected ^hm dC derivative (Fig. 2 , 3 ) was then benzylated, tritylated and phosphytylated using established methods ( 17 , 18 ). The ^hm C-phosphoramidite ( 4 ) prepared by this method was incorporated into synthetic oligonucleotides with efficiency indistinguishable from normal bases.

Figure 2 . Synthetic scheme for the preparation of the ^hm C phosphoramidite. ( a ) cyanoethyl alcohol/trifluoroacetic acid; ( b ) acetic anhydride/pyridine; ( c ) triazole/phosphoryl chloride/triethylamine/acetonitrile; ( d ) dioxane/aqueous ammonia; ( e ) trimethylsilyl chloride/pyridine; ( f ) benzoyl chloride/ pyridine/ammonia; ( g ) 4,4'-dimethoxytrityl chloride/DMAP/pyridine; ( h ) cyanoethyl- N , N -diisopropylchlorophosphoramidite/ diisopropylamine/acetonitrile; (i) concentrated aqueous ammonia/60^oC.

Because ^hm dC and dC are difficult to separate by HPLC, we first prepared a 12-base test sequence containing ^hm dC, dT, dG and dA: 5' (T G ^hm C A) ₃ .

The ^hm C-containing oligonucleotide was deblocked by treatment in aqueous ammonia at 60^oC for 60 h and purified by HPLC. Although 12 h is sufficient to remove the cyanoethyl protecting group from the monomer, prolonged treatment is required to remove the protecting group from the protected residue in the oligonucleotide. A portion of the ^hm C-containing oligonucleotide was enzymatically digested with nuclease P1 and bacterial alkaline phosphatase. The liberated deoxynucleosides were then analyzed by HPLC using a reverse phase column.

The chromatogram shown in Figure 3 shows four predominant peaks corresponding to the three normal deoxynucleosides and ^hm dC as well as a minor peak eluting at 9.6 min. The ^hm dC peak was identified by co-chromatography with an authentic standard. The UV spectrum of the ^hm dC peak, obtained with the diode array detector (Fig. 4 , top), shows absorbance maxima and minima of 273 nm and 251 nm respectively, consistent with reported values ( 26 ).

Figure 3 . HPLC chromatogram of the deoxynucleosides obtained following enzymatic digestion of an oligonucleotide containing dT, dG, dA and ^hm dC. The small peak at 9.6 min is protected ^hm dC.

Figure 4 . Characterization of ^hm dC from the synthetic oligodeoxynucleotide. ( Top ) UV spectrum of the ^hm dC peak obtained with the diode array detector. ( Bottom ) Mass spectrum of the silylated ^hm C peak derived from the HPLC chromatogram shown in Figure 3.

The ^hm dC obtained by HPLC chromatography was dried, silylated and analyzed by GC/MS. The mass spectrum of the silylated ^hm C peak at 13.7 min is shown in Figure 4 (lower). The most abundant ion is the parent ion at m/z 357. Additional ions resulting from fragmentation of the 5-substituent are identified in Figure 4 (lower).

The minor peak in the HPLC chromatogram (Fig. 3 ) eluting at 9.6 min is the cyanoethyl-protected ^hm dC derivative (Fig. 2 , 3 ). Based upon integration of the chromatogram peaks, deprotection of the ^hm C residue was ~96% complete. No peak corresponding to ^hm dU, which could result from hydrolytic deamination of ^hm dC, was observed. Treatment of the oligonucleotide with sodium methoxide overnight at room temperature eliminated residual traces of the cyanoethyl-protected ^hm dC.

Having established the synthetic method for placement of ^hm C residues, we prepared a series of longer oligonucleotides containing cytosine, 5 ^m C or ^hm C at selected sites (Table 1 ). The sequence of the oligonucleotide contains the recognition site for the Msp I/ Hpa II restriction endonucleases. Following deprotection and purification, the composition of the oligonucleotides was examined by GC/MS following acid hydrolysis in 60% formic acid. Data was collected in the selected ion mode using the most abundant ions of each of the bases in the oligonucleotides. The results are shown in Figure 5 .

Figure 5 . Sum of the selected ion chromatograms (parent ions) of oligodeoxynucleotide hydrosylates containing (top) all normal bases, (center) an oligonucleotide in which one cytosine has been replaced by 5 ^m C, and (lower) an oligonucleotide in which one cytosine has been replaced by ^hm C. The base sequence corresponds to the upper strand (Materials and Methods).

An oligonucleotide corresponding to the upper strand (Materials and Methods), containing only A,T, G and C generated four chromatographic peaks identified by their characteristic retention times and mass spectra (Fig. 5 , upper). An oligonucleotide of the same sequence except for the replacement of the inner cytosine of the Msp I/ Hpa II cut site with 5 ^m C (Table 1 , Duplex 2) was prepared and analyzed in an identical fashion. The silylated 5 ^m C peak is observed at 11.3 min (Fig. 5 , center). An additional oligonucleotide was then generated in which the same inner cytosine residue was replaced by ^hm C. The ^hm C peak is observed at 13.7 min (Fig. 5 , lower). As reported previously for the analysis of ^hm U in oligonucleotides ( 11 , 12 ), we also observe that acid hydrolysis results in considerable destruction of ^hm C as indicated by the reduced intensity of the ^hm C peak in Figure 5 (lower). The size of the observed ^hm C chromatographic peak is consistent with the expected amount of ^hm C in the oligonucleotide based upon results obtained following acid hydrolysis of standards. The selected ion chromatogram corresponding to the most abundant ion of the silylated ^hm C derivative ( m/z 357) is shown in the inset of Figure 5 (lower).

The oligonucleotides were combined with cytosine-containing complementary sequences to generate a series of duplexes with asymmetrically modified cytosine residues at either the inner or outer cytosine residue of the C/CGG recognition sequence. Complementary strands were also synthesized with 5 ^m C to generate symmetrically modified recognition sites (Table 1 ). Oligonucleotide duplexes, ³² P-labelled in both strands, were incubated with either Msp I or Hpa II for 15 and 30 min respectively. The resulting oligonucleotide fragments were then separated by electrophoresis on a 20% polyacrylamide denaturing gel and analyzed by a PhosphorImager. The results obtained are shown in Figure 6 .

Figure 6 . Cleavage of synthetic oligonucleotides by restriction endonucleases: ( Top ) Hpa II, ( Bottom ) Msp I. Lane markers 1 through 9 correspond to the duplexes shown in Table 1.

Previously it had been reported that ^hm C-containing bacteriophage DNA is not cleaved by either Msp I or Hpa II ( 27 ). In the bacteriophage DNA, however, both cytosine residues of the recognition site, in both strands are replaced by ^hm C. Only by chemical synthesis is it possible to selectively modify one of the cytosine residues. We observe that the duplex containing ^hm C at the inner position (Table 1 , Duplex 3; Fig. 6 , lane 3) is cleaved by Msp I, however, if the ^hm C residue is placed at the outer cytosine position (Table 1 , Duplex 5; Fig. 6 , lane 5), the modified strand is protected from Msp I cleavage whereas the unmodified strand is cleaved. The same pattern is observed when the ^hm C residue is replaced by 5 ^m C. The only difference observed between ^hm C and 5 ^m C is that 5 ^m C at the outer position (Table 1 , Duplex 4; Fig. 6 , lane 4) completely protects the modified strand from Msp I cleavage whereas the ^hm C-containing strand (lane 5) is cleaved to a small extent. Placement of either 5 ^m C or ^hm C at either position completely protects the modified strand from cleavage by Hpa II and the cleavage efficiency of the unmodified complementary strand is reduced. Previously, it has been reported that the placement of 5 ^m C within the Msp I/ Hpa II recognition site selectively protects only the modified strand of the duplex from cleavage ( 28 ). The similarity in results obtained with 5 ^m C and ^hm C-containing oligonucleotides with these restriction endonucleases provides additional support of the validity of the method reported here for placement of ^hm C residues in oligodeoxynucleotides.

DISCUSSION

We report here a method for the synthesis of oligodeoxynucleotides containing ^hm C residues at selected sites. This strategy is based upon the selective reactivity of the 5-hydroxymethyl group of ^hm dU with alcohols under acidic conditions. The cyanoethyl ether protecting group generated is stable toward ammonia at room temperature, allowing conversion from the protected uracil derivative to the protected cytosine derivative. At elevated temperatures in aqueous ammonia, the cyanoethyl ether is cleaved by [beta]-elimination. Prolonged ammonolysis is required to remove the protecting group from the oligonucleotide which would prevent simultaneous use of the protected ^hm C derivative reported here with alkaline-labile modified bases.

The base composition of the oligonucleotides prepared by this method has been verified by both enzymatic digestion and HPLC analysis as well as acid hydrolysis and GC/MS. The validity of this synthetic method is further supported by studies with methylation-sensitive restriction nucleases in which it is demonstrated that the placement of ^hm C and 5 ^m C similarly protect oligodeoxynucleotides from cleavage.

The mechanism by which cytosine methylation influences gene control is as yet unknown, however, it is suspected that such an effect may be modulated through sequence specific DNA-protein interactions sensitive to the presence of the methyl group. Oxidation of 5 ^m C to ^hm C could impact DNA-protein interactions because replacement of the hydrophobic methyl group by the hydrophilic hydroxymethyl group may prevent or alter the binding of proteins having hydrophobic binding clefts for the 5 ^m C methyl group. Recently, we demonstrated that replacement of T by ^hm U residues increases the dissociation rate of restriction nucleases from the cleaved substrates ( 29 ).

A mechanism by which 5 ^m C oxidation could have a heritable effect upon methylation patterns is by blocking methyl-directed methylation following DNA replication. It is known that hemimethylated DNA, generated during DNA replication, is a substrate for cytosine methyltransferase and that the cytosine 5-methyl group in the parental strand directs the enzymatic methylation of the complementary progeny strand. Smith and coworkers have previously shown that the ability of cytosine 5-substituents to direct methylation of the complementary strand is a function of the size of the substituent ( 30 ). A sharp maximum was seen at the size of the methyl group. Oxidation from methyl to hydroxymethyl not only increases the size of the substituent significantly, but the hydrophilic hydroxyl group is likely well hydrated and may not be accommodated within a binding cleft designed to hold a hydrophobic methyl group. If unrepaired, oxidation of the 5-methyl group could then prevent maintenance methylation. The influence of ^hm C on the methylase is as yet unknown, however, such studies are currently in progress.

ACKNOWLEDGEMENTS

We wish to acknowledge support from the National Institutes of Health (GM-41336, GM-50351, CA-33572) and the National Science Foundation (BIR-9220534) which provided funds for the PhosphorImager. Special thanks are extended to Dr John Termini for providing the laboratory facilities needed to perform experiments with labelled oligonucleotides.

REFERENCES

1 Wyatt, G.R. and Cohen, S.S. (1953) Biochem. J. 55, 774-782.

2 Shapiro, H.S. (1970) In Sober, H.H. (ed.) Handbook of Biochemistry, CRC Press, Cleveland.

3 Cier, A., Lefier, A., Ravier, M. and Nofre,C. (1962) C.R. Hebd. Seances Acad. Sci. 254, 504-506.

4 Khattak, M.N. and Green, J.H. (1966) Int. J. Radiat. Biol. 11, 137-143.

5 Privat, E. and Sowers, L.C. (1996) Chem. Res. Tox. 9, 745-750.

6 Steinberg, J.J., Cajigas, A. and Brownlee, M. (1992) J. Chromatogr. 574, 41-55. MEDLINE Abstract

7 Cannon, S.V., Cummings, A. and Teebor, G.W. (1988) Biochem. Biophys. Res. Commun. 151, 1173-1179. MEDLINE Abstract

8 Razin, A. and Riggs, A.D. (1980) Science 210, 604-610. MEDLINE Abstract

9 Jones, P.A., Rideout (III), W.M., Shen, J.-C., Spruck, C.H. and Tsai, Y.C. (1992) BioEssays 14, 33-36. MEDLINE Abstract

10 Laird, P.W. and Jaenisch, R. (1994) Hum. Mol. Genet. 3, 1487-1495. MEDLINE Abstract

11 Frenkel, K., Zhong, Z., Wei, H., Karkoszka, J., Patel, U., Rashid, K., Georgescu, M. and Solomon, J.J. (1991) Anal. Biochem. 196, 126-136. MEDLINE Abstract

12 Cadet,J., Incardona,M.-F., Odin, F., Molka, D., Mouret, J.-F., Poiverilli, M., Faure, H., Ducros, V., Tripier, M., and Favier, A. (1993) IARC 124, 271-276.

13 Mullart, E., Lohman, P.H.M., Berends, F. and Vijg,J. (1990) Mutation Res. 237, 189-210.

14 Masuda, T., Shinohara, H., and Kondo, M. (1975) J. Radiat. Res. 16, 153-161. MEDLINE Abstract

15 Shiau, G.T., Schinazi,R.F., Chen, M.S., and Prusoff, W.H. (1980) J. Med. Chem. 23, 127-133. MEDLINE Abstract

16 Divakar, K.J. and Reese, C. B. (1982) J. Chem. Soc. Perkin 1, 1982, 1171-1176.

17 Ti,G.S., Gaffney,B.L. and Jones,R.A. (1982) J. Am. Chem. Soc., 104, 1316-1319.

18 Gait, M.J. (1984) Oligonucleotide Synthesis, A Practical Approach, IRL Press, Oxford, UK.

19 Kasai,H., Crain,P.F., Kuchino,Y., Nishimura,S., Ootsuyama,A. and Tanooka,H. (1986) Carcinogenesis, 7, 1849-1851. MEDLINE Abstract

20 Dizdaroglu, M. (1984) J. Chromatogr. 295, 103-121. MEDLINE Abstract

21 Sowers, L.C. and Beardsley, G.P (1993) J. Org. Chem. 58, 1664-1665.

22 Xu, Y.-Z., Zheng, Q., and Swann, P.F. (1992) J. Org. Chem. 57, 3839-3845.

23 Cline, R.E., Fink, R.M. and Fink, K. (1959) J. Am. Chem. Soc. 81, 2521-2527.

24 Scheit, K.H. (1966) Chem. Ber. 99, 3884-3890.

25 Christopherson, M.S., and Broom, A.D. (1991) Nucleic Acids Res. 19, 5719-5724. MEDLINE Abstract

26 Loeb, M.R. and Cohen, S.S. (1959) J. Biol. Chem. 234, 364-369.

27 Huang, L.-H., Farnet,C.M., Ehrlich, K.C., and Ehrlich, M. (1982) Nucleic Acids Res. 10, 1579-1591. MEDLINE Abstract

28 Butkus, V, Petrauskiene, L., Maneliene, Klimasauskas, S., Laucys, V., and Januiatis, A. (1987) Nucleic Acids Res. 15, 7091-7102. MEDLINE Abstract

29 Mazurek, M., and Sowers, L.C. (1996) Biochemistry 35, 11522-11528. MEDLINE Abstract

30 Hardy,T.A., Baker,D.J., Newman, E.M., Sowers, L.C., Goodman, M.F., and Smith, S.S. (1987) Biochem. Biophys. Res. Comm. 145, 146-152. MEDLINE Abstract

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^* To whom correspondence should be addressed. Tel: +1 818 359 8111; Fax: +1 818 301 8458; Email: lsowers@smtplink.coh.org Present addresses: ⁺ Ecole Nationale Supérieure de Chimie de Rennes, France and ^[dagger] Massachusetts Institute of Technology, Cambridge, MA, USA
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