Base-pairing properties of N4-methoxydeoxycytidine 5'-triphosphate during DNA synthesis on natural templates, catalyzed by DNA polymerase I of Escherichia coli

Publisher Website
Google Scholar
Abstract
N4-Methoxydeoxycytidine 5''-triphosphate (mo4dCTP) was synthesized by reaction of dCTP with methoxyamine and then purified by high-performance liquid chromatography (HPLC) and used to analyze the specificity of mo4dCMP incorporation during polymerization on natural templates, catalyzed by DNA polymerase I of E. coli. Elongation of synthetic 5''-32P-labeled primers, annealed to single-stranded DNA of bacteriophage M13, was carried out in the presence of only 3 of the 4 normal dNTPs; then, reaction products were displayed by high-resolution gel electrophoresis and visualized by autoradiography. By measuring primer elongation in each of the 4 minus reactions with and without added mo4dCTP, the specificity of mo4dCMP incorporation were examined at different positions along the M13 template. mo4dCTP apparently is utilized most readily (although at low efficiency) in place of dTTP during DNA synthesis, the analog can also replace dCTP during primer elongation, although at barely detectable efficiency, and the ease at which both mo4C .cntdot. A and mo4C .cntdot. G pairs are formed during DNA synthesis on natural templates is markedly influenced by the nucleotide sequence of the template.