Specificity and Function of Exonuclease II in the Synthesis of Alternating Copolymers by DNA Polymerase

The poly d(A‐T) dependent synthesis of the analog alternating copolymer poly d(A‐4thioT) (in which thymidine is replaced by 4‐thiothymidine) by DNA polymerase of Escherichia coli is accompanied by hydrolysis of at least one mole of primer d(A‐T) per mole of product d(A‐4thioT) formed. Exonuclease II, the deoxyribonuclease associated with DNA polymerase, is responsible for the hydrolysis of the primer. Since the growing 3′ end of the primer is protected from hydrolysis during the enzymatic polymerisation the hydrolysis must occur at the free 5′ end. The enzymatic synthesis of poly d(A‐4thioT) on a poly d(A‐T) template proceeds in a two phase time course. The ratio of polymerisation versus hydrolysis is close to unity for the first rapid phase of synthesis. The temperature‐dependence of poly d(A‐T) and poly d(A‐4thioT) synthesis, represented by usual Arrhenius plots has a discontinuity at 15° and 25°, respectively. Exonuclease II exhibits a similar discontinuity at 26° under the same conditions. Above 25°, the synthesis of poly d(A‐4thioT) has a smaller apparent activation energy than the poly d(A‐T) synthesis, but the latter is twice to three times as fast. The obligatory hydrolysis of the primer offers a plausible explanation of why the synthesis of poly d(A‐4thioT) depends on the amount of poly d(A‐T) primer in a stoichiometric manner since the former copolymer is not a primer for extensive synthesis of poly d(A‐4thioT). A modified enzyme model for DNA polymerase is suggested, implicating the function of exonuclease II in providing an additional driving force for the unidirectional movement of the enzyme along the template. Emphasis is put on the energy contribution arising from formation of new base pairs during synthesis. The specificity of exonuclease II was also studied using as substrates such polymers which consist of poly d(A‐T) and poly d(A‐4thioT) in one half each. The polymer bearing the d(A‐4thioT) n at the 3′ half is readily hydrolysed by exonuclease II from both the 3′ and 5′ end synchronously at pH 7.2. Upon mercuration of d(A‐4thioT) n with p ‐chloromercuribenzoate, preferentially the poly d(A‐T) part is hydrolysed from the 5′ end at a reduced rate. In contrast, no synchronism of nuclease reaction is observed with the polymer bearing d(A‐4thioT) n at the 5′ end. the latter being generally more resistant against hydrolytic attack by the enzyme.