An active site aromatic triad in Escherichia coli DNA Pol IV coordinates cell survival and mutagenesis in different DNA damaging agents.

Ryan W Benson,Veronica G Godoy,Ida Lin,Matthew D Norton,William S Du Comb

doi:10.1371/journal.pone.0019944

Abstract

DinB (DNA Pol IV) is a translesion (TLS) DNA polymerase, which inserts a nucleotide opposite an otherwise replication-stalling N2-dG lesion in vitro, and confers resistance to nitrofurazone (NFZ), a compound that forms these lesions in vivo. DinB is also known to be part of the cellular response to alkylation DNA damage. Yet it is not known if DinB active site residues, in addition to aminoacids involved in DNA synthesis, are critical in alkylation lesion bypass. It is also unclear which active site aminoacids, if any, might modulate DinB's bypass fidelity of distinct lesions. Here we report that along with the classical catalytic residues, an active site “aromatic triad”, namely residues F12, F13, and Y79, is critical for cell survival in the presence of the alkylating agent methyl methanesulfonate (MMS). Strains expressing dinB alleles with single point mutations in the aromatic triad survive poorly in MMS. Remarkably, these strains show fewer MMS- than NFZ-induced mutants, suggesting that the aromatic triad, in addition to its role in TLS, modulates DinB's accuracy in bypassing distinct lesions. The high bypass fidelity of prevalent alkylation lesions is evident even when the DinB active site performs error-prone NFZ-induced lesion bypass. The analyses carried out with the active site aromatic triad suggest that the DinB active site residues are poised to proficiently bypass distinctive DNA lesions, yet they are also malleable so that the accuracy of the bypass is lesion-dependent.

Highlights

Replicative DNA polymerases are multi-protein complexes responsible for synthesizing a high fidelity copy of a cell’s genome
DinB active site residues are important for survival in methyl methanesulfonate (MMS)
The aromatic triad is required for in vivo DinB TLS In an effort to gain insights into the TLS activity of DinB in alkylation lesion bypass, we looked for conserved residues in the DinB active site that could be as important as F13 in DinB N2 group of deoxyguanosine (N2-dG) TLS

Summary

Introduction

Replicative DNA polymerases are multi-protein complexes responsible for synthesizing a high fidelity copy of a cell’s genome. To avoid lethality, specialized DNA polymerases insert deoxynucleotides (dNTPs) opposite replication-blocking DNA lesions in a process known as translesion synthesis (TLS). This is largely a low fidelity process usually resulting in elevated mutagenesis [1,2]. DinB is of particular interest because of its evolutionary conservation [1,6,8] and its high basal intracellular concentration (,250 nM) [1,9,10] This is approximately 17 fold higher [10] than that of DNA Pol III complex (the replicative DNA polymerase, 15 nM; [9]) and is similar to that of the processivity clamp (b-clamp, 250 nM; [11,12]), an essential replication factor known to both recruit all DNA polymerases to the replication fork and manage their activity in the cell [13,14]

Methods

Results

Conclusion