O4-Methyldeoxythymidine replacing deoxythymidine in poly[d(A—T)] renders the polymer resistant to the 3′ → 5′ exonuclease activity of the Klenow and T4DNA polymerases

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Abstract
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 α-phosphorothionate linkage did not prevent measurable digestion of polytd(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.

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