Escherichia coli polymerase I can use O2-methyldeoxythymidine or O4-methyldeoxythymidine in place of deoxythymidine in primed poly(dA-dT).poly(dA-dT) synthesis.

Abstract

O2- and O4-alkyldeoxythymidine are among the 4 O-alkyl base-modified derivatives produced by the reaction of N-nitroso alkylating agents with nucleic acids in vitro and in vivo. O2- and O4-methyl-dTTP can substitute for dTTP in alternating poly(dA-dT)-primed DNA synthesis. Up to 22% of the pyrimidines in these newly synthesized polymer were found by HPLC [high performance liuqid chromatography] analysis to be O-methyldeoxythymidine. Little polymer synthesis was observed in the absence of dTTP. The O-methyl-dTTP did not inhibit polymerization of daTP and dTTP. Polymers containing O2- or O4-methyldeoxythymidine were obtained in good yield, retaining the secondary structure of alternating poly(dA-dT). This was shown by the data for thermal transition under different conditions. Poly(dA-dT) .cntdot. poly(dA-dT) methylated or ethylated to less than 4% total modification by alkylnitrosoureas had a distinctly less stable structure. Neither O2- nor O4-methyldeoxythymidine can form more than H bond with adenosine. The unchanged secondary structure of polymers containing these modified thymidines indicates that stacking interactions must play a major role in helix stabilization. O-Alkyldeoxythymidine may be formed by N-nitroso carcinogens that react intracellularly. The triphosphates can be utilized by E. coli DNA polymerase I as dTTP. The incorporated O4-methyl-dT causes misincorporation of G in transcription and synthesis. When O2-methyl-dT is present, less, but definite, misincorporation results.