C4-methyldeoxythymidine replacing deoxythymidine in poly[d(A-T)] renders the polymer resistant to the 3'----5' exonuclease activity of the Klenow and T4 DNA polymerases.
We previously reported that O4-alkyl dTTPs could replace, for short times, dTTP in polymer synthesis [Singer et al., PNAS 83, 26-32, 1986]. The reasons for such early termination of synthesis could be either proofreading or the eventual formation of weakly paired primer termini. Utilizing the known 3'----5' exonucleolytic activity of polymerases, in the absence of dNTPs, enabled us to conclude that, in contrast to the digestibility of poly[d(A-T)] which yielded the expected 3'-mononucleotides, the polymerizing enzymes did not digest O4-methyl dT or its neighbors. The presence of the resistant alpha-phosphorothionate linkage did not prevent measurable digestion of poly[d(A-T)] by the Klenow fragment. This, together with evidence that polymerization of O4-methyl dTTP is favored at low temperatures, supports the model proposed by Ollis et al. [Nature 313, 762-766, 1985] showing independent domains for the two activities in the Klenow fragment.
D000478:Alkylation; D004256:DNA Polymerase I / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D007700:Kinetics; D013379:Substrate Specificity; D013604:T-Phages / Q000201:enzymology; D013936:Thymidine / Q000031:analogs & derivatives*
C008505:O-methylthymidine; D004256:DNA Polymerase I; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; D013936:Thymidine