TEMPLATE-PRIMER-DEPENDENT TURNOVER OF (SP)-DATP-ALPHA-S BY T4 DNA-POLYMERASE - THE STEREOCHEMISTRY OF THE ASSOCIATED 3'-]5'-EXONUCLEASE

Abstract

T4 DNA polymerase converts (Sp)-2''-deoxyadenosine 5''-O-(1-thio[1-18O2]triphosphate) to 2''-deoxyadenosine 5''-O-[18O]-phosphorothioate in the presence of poly(d(A-T).cntdot.poly(d(A-T)) template-primer. Control experiments involving either omitting the poly(d(A-T).cntdot.poly(d(A-T)) template-primer or employing the (Rp)-2''-deoxyadenosine 5''-O-(1-thiotriphosphate) diastereomer showed no reaction. This conversion, as in the P-O case, apparently involves incorporation of the thionucleotide into the poly(d(A-T) followed by hydrolysis resulting from the 3'' .fwdarw. 5''-exonuclease activity. The 2''-deoxyadenosine 5''-O-[18O]phosphorothioate was converted to (Sp)-2''-deoxyadenosine 5''-O-(1-thio[1-18O]triphosphate), with no change in the configuration at P.alpha. by using the coupled adenylate kinase-pyruvate kinase enzyme system. A 31P NMR spectrum of the product showed that the 18O was entirely in the nonbridging position, indicating an overall retention in the net turnover process (i.e., incorporation followed by excision). Since the incorporation process involves an inversion of configuration around the phosphorus, it must be inferred that the 3'' .fwdarw. 5''-exonuclease activity of T4 polymerase proceeds with inversion of configuration at the phosphorus atom, most simply via a direct displacement mechanism. This finding represents the 1st example of phosphodiester hydrolysis catalyzed by an exonuclease that does not involve a covalent phosphoryl-enzyme intermediate.