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

Abstract

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 a t least one mole of primer d(A-T) per mole of product d(A-4thioT) formed. Exonuclease 11, 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.ExonucIease I1 exhibits a similar discontinuity a t 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(A4thioT). A modified enzyme model for DNA polymerase is suggested, implicating the function of exonuclease I1 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 I1 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), a t the 3' half is readily hydrolysed by exonuclease I1 from both the 3' and 5' end synchronously at pH 7.2. Upon mercuration of d(A-4thioT), with p-chloromercuribenzoate, preferentially the poly d(A-T) part is hydrolysed from the 5' end at a reduced rate. I n contrast, no synchronism of nuclease reaction is observed with the polymer bearing d(A-4thioT), at the 6' end, the latter being generally more resistant against hydrolytic attack by the enzyme. Enzymes. DNA polymerase or deoxynucleosidetriphosphate: DNA deoxynucleotidyl-transferase (EC 2.7.7.7) ; thymidine kinase (EC 2.7.1.21); thymidine monophosphate kinase (EC 2.7.4.9).feature of a nucleotide analog not present in DNA from natural sources could not be explained and subsequent studies were pursued with the aim of obtaining more insight into the enzymatic mechanism of a template-directed polymerisation in this model system. Increasing evidence for an important role of deoxyribonucleases in the process of DNA replication, as reviewed by Lehmann f2], and the surprising discovery of Klett et at.[3] that exonuclease I1 can hydrolyse poly d(A-T) from the ...

Synthesis of Poly[d(A‐s4T) · d(A‐s4T)] by Bacillus subtilis DNA Polymerase

“…Table 1. This reduction in rate is similar to that observed earlier [4] with DNA polymerase of E . coli.…”

“…coli. The important finding of Klenow and Henningsen [9] that the 5' exonuclease activity can be removed -d(A-T)] as reported earlier [4]. Therefore, one essential difference of DNA polymerase from B. subtilis is that it can synthesize the analogous polymer de novo and extensively.…”

Biosynthesis of DNA

“…The stoichiometric requirement for poly d(A-T ) in the synthesis of poly d(A-4-thio-T) (57) has led to the suggestion that 5'�3' hydrolysis may actually provide additional driving force for the linear movement of the enzyme along the template (58). However, against this is the fact that there is actually partial inhibition of synthesis by apposition of 5' termini to 3' termini in the poly dA· oligo dT system, in contrast to the observed stimu lation of 5'�3' hydrolysis.…”