Resolving a fidelity paradox: why Escherichia coli DNA polymerase II makes more base substitution errors in AT- compared with GC-rich DNA.

Zhijie Wang,Eli Lazarov,Mike O'Donnell,Myron F Goodman

doi:10.1074/jbc.m110006200

Abstract

The activity of DNA polymerase-associated proofreading 3'-exonucleases is generally enhanced in less stable DNA regions leading to a reduction in base substitution error frequencies in AT- versus GC-rich sequences. Unexpectedly, however, the opposite result was found for Escherichia coli DNA polymerase II (pol II). Nucleotide misincorporation frequencies for pol II were found to be 3-5-fold higher in AT- compared with GC-rich DNA, both in the presence and absence of polymerase processivity subunits, beta dimer and gamma complex. In contrast, E. coli pol III holoenzyme, behaving "as expected," exhibited 3-5-fold lower misincorporation frequencies in AT-rich DNA. A reduction in fidelity in AT-rich regions occurred for pol II despite having an associated 3'-exonuclease proofreading activity that preferentially degrades AT-rich compared with GC-rich DNA primer-template in the absence of DNA synthesis. Concomitant with a reduction in fidelity, pol II polymerization efficiencies were 2-6-fold higher in AT-rich DNA, depending on sequence context. Pol II paradoxical fidelity behavior can be accounted for by the enzyme's preference for forward polymerization in AT-rich sequences. The more efficient polymerization suppresses proofreading thereby causing a significant increase in base substitution error rates in AT-rich regions.

Highlights

The activity of DNA polymerase-associated proofreading 3؅-exonucleases is generally enhanced in less stable DNA regions leading to a reduction in base substitution error frequencies in AT- versus GC-rich sequences
A reduction in fidelity in AT-rich regions occurred for polymerase II (pol II) despite having an associated 3؅-exonuclease proofreading activity that preferentially degrades ATrich compared with GC-rich DNA primer-template in the absence of DNA synthesis
The main goal of these experiments was to determine the effects of sequence context on the fidelity of DNA synthesis by proofreading proficient E. coli pol II HE

Summary

Introduction

The activity of DNA polymerase-associated proofreading 3؅-exonucleases is generally enhanced in less stable DNA regions leading to a reduction in base substitution error frequencies in AT- versus GC-rich sequences. A reduction in fidelity in AT-rich regions occurred for pol II despite having an associated 3؅-exonuclease proofreading activity that preferentially degrades ATrich compared with GC-rich DNA primer-template in the absence of DNA synthesis. An important corollary is that mismatches are less susceptible to proofreading in relatively stable GC-rich regions of DNA, leading to base substitution hot spots, whereas the opposite is true in AT-rich DNA [13]. Studies with bacteriophage T4 pol support this model by demonstrating that nucleotide substitution errors tend to be lower in surroundings rich in AT base pairs because proofreading is favored in the less stable AT-rich regions [13,14,15,16]. Pol II was found to copy several types of DNA damage in vivo [21, 22] and to moderate the level of adaptive mutation in nondividing cells [23] catalyzed primarily by error-prone pol IV [24, 25]

Methods

Results

Conclusion