ENZYMIC SYNTHESIS OF DNA .33. HYDROLYSIS OF A 5'-TRIPHOSPHATE-TERMINATED POLYNUCLEOTIDE IN ACTIVE CENTER OF DNA POLYMERASE

Detailed analysis of how DNA polymerase degrades a DNA chain from the 5′ end (5′ → 3′ hydrolysis) has contributed to a comprehensive model of the active site and enzymic mechanism. 5′ → 3′ hydrolysis requires that the substrate chain be part of a duplex and up to 20% of the cleavages are at a subterminal diester bond. These properties do not apply to 3′ → 5′ hydrolysis by DNA polymerase. With a 5′-triphosphate-terminated polythymidylate (pppT(pT)300)∥ ∥ Abbreviations used: TB, triethylammonium bicarbonate; A, G, C and T for the deoxyribonucleosides or deoxyribonucleotides of adenine, guanine, cytosine and thymine; ddT for 2′, 3′-dideoxythymidine; poly dX or d(X)n for a homopolymer; pppT(pT)n for a 5′-triphosphate-terminated poly dT; pppT(pT)npddT for 5′-triphosphate-and 3′-dideoxythymidine-terminated poly dT. as substrate for 5′ → 3′ hydrolysis, the initial cleavage produces principally (about 75%) the dinucleoside tetraphosphate, pppTpT. With a 5′-monophosphate-terminated polythymidylate, pT(pT)300, the principal cleavage product (about 75%) is the mononucleotide, pT. The preference for cleavage at the penultimate rather than terminal diester bond of pppT(pT)300 is attributed to binding of the 5′-triphosphate terminus in the enzyme site ordinarily occupied by the deoxyribonucleoside triphosphate, pppT, during polymerization. This result suggests the proximity of the 5′ → 3′ exonuclease site to the polymerizing site in the active center of the enzyme. The influence of a neighboring 3′ hydroxyl terminus, in a nicked region of a DNA duplex, on the rate and character of the 5′ → 3′ cleavage supports this conclusion. The DNA polymerase induced by phage T4 infection lacks the 5′ → 3′ exonuclease function. Neither the Escherichia coli nor the T4 polymerase can direct any detectable synthesis in the 3′ → 5′ direction as judged by the utilization of the 5′-triphosphate-terminated chain as a primer. Synthesis with both enzymes is exclusively in the 5′ → 3′ direction. © 1969.