Studies on the mechanism of Escherichia coli DNA polymerase I large fragment. Effect of template sequence and substrate variation on termination of synthesis.

J Abbotts,D N Sengupta,G Zon,S H Wilson

doi:10.1016/s0021-9258(18)68150-5

Abstract

Termination of Escherichia coli DNA polymerase I large fragment after processive synthesis on natural and other well-defined template.primer systems has been examined. We found that after any given deoxynucleoside monophosphate incorporation termination occurs in a nonrandom manner with phi X174 DNA as template: Termination is much more likely at some nucleotide residues along the template than at others. Analysis of these stronger termination sites indicates that the template base:incoming nucleotide combination influences termination. Introduction of a double-stranded region along the phi X174 template induces termination, and reducing dNTP concentrations or substituting 2'-deoxynucleoside 5'-O-(1-thio)triphosphate substrates also increases termination. Observations with the phi X174 DNA template system were extended with a defined template containing 1 inosine residue in an otherwise d(T)n homopolymer. Termination at the I residue is modulated by dCTP and decreases as dCTP concentration increases. A similar relationship is seen with the dCTP (1-thio) derivative, but termination is higher at given concentrations of this derivative than with dCTP. Pyrophosphate decreases general processivity in this system, but does not counteract the effect of increasing dCTP. Hill plot analysis of the dCTP effect in the inosine-containing template system gave a linear plot with Hill coefficient of 0.34, suggesting that dCTP influences termination at several steps in the polymerase reaction scheme. Substituting a methylated template base for I also increased termination, producing very strong blocks to processive synthesis. The results are consistent with a model in which termination occurs with several enzyme forms that are in equilibrium in an ordered catalytic mechanism.

Highlights

Termination of Escherichia coli DNA polymerase I primer and incorporates many dNMP residues before dissolarge fragment after processive synthesis on natural ciating from the template.nascent chaincomplex
This stems both from lack of appropriate assays for termination and lack of a theoretical framework within which to study termination.In thepresent study, we address both of these problems in that several ways of experimentally modulating chain termination are described, and theresults are discussed in the context of a hypothetical scheme involving an ordered mechanism with four enzyme forms capable of termination
Termination of Synthesis by Pol I If lf as a function of template nucleotide sequence, instead of secondary structure, we conducted experiments where the template was a region of 4x174 DNA, residue 652 to residue 550 of the plus strand, thaits secondary structure-free on the basis of computer-derived predictions and previous enzymatic studies [10]

Summary

Introduction

Termination of Escherichia coli DNA polymerase I primer and incorporates many dNMP residues before dissolarge fragment after processive synthesis on natural ciating from the template.nascent chaincomplex. Analysis of the mechanism of product release and chain termination after processive synthesis has lagged behind analysis of the initiation phase of synthesis Analysis of these stronger termination sites indicates For example, insight into whether termination is ordered that the template base:incoming nucleotide combina- uersus random or to the number of enzyme forms capable of tion influences termination. Earlier reports on DNA polymerase termination involved relationship isseen with the dCTP (1-thio) derivative, homopolymer replication systems and incubation conditions but termination is higher at given concentrations of where each product molecule represented only one chain this derivative than with dCTP. Measurement of the number of product molecules, and chain termination events, corresponding to each dNMP addition along a templaterevealed that termination is influenced by a complicated array of variables It was possible, to identify general features at several steps in the polymerase reaction scheme. Third,the chance for termination is higher after addition of the first several dNMP residues to a primer than after addition of more than 7 residues, suggestingsome change in the enzymatic mechanism as synthesis proceeds from initiation toprocessive elongation [2]

Methods

Results

Conclusion