Abstract

The fidelity of DNA replication requires an appropriate balance of dNTPs, yet the nascent leading and lagging strands of the nuclear genome are primarily synthesized by replicases that differ in subunit composition, protein partnerships and biochemical properties, including fidelity. These facts pose the question of whether imbalanced dNTP pools differentially influence leading and lagging strand replication fidelity. Here we test this possibility by examining strand-specific replication infidelity driven by a mutation in yeast ribonucleotide reductase, rnr1-Y285A, that leads to elevated dTTP and dCTP concentrations. The results for the CAN1 mutational reporter gene present in opposite orientations in the genome reveal that the rates, and surprisingly even the sequence contexts, of replication errors are remarkably similar for leading and lagging strand synthesis. Moreover, while many mismatches driven by the dNTP pool imbalance are efficiently corrected by mismatch repair, others are repaired less efficiently, especially those in sequence contexts suggesting reduced proofreading due to increased mismatch extension driven by the high dTTP and dCTP concentrations. Thus the two DNA strands of the nuclear genome are at similar risk of mutations resulting from this dNTP pool imbalance, and this risk is not completely suppressed even when both major replication error correction mechanisms are genetically intact.

Highlights

  • The integrity of an organism’s genome is vital to its continued survival, whether unicellular microbe or complex large mammal [1]

  • We find that the leading and lagging strand replication fidelity is affected by the dNTP pool imbalance and that the mismatch repair machinery corrects replication errors driven by a dNTP pool imbalance with highly variable efficiencies

  • Results dNTP pools of the rnr1-Y285A and msh2D strains To examine potential differences in mutational specificity between the major replicative polymerases in the presence of the imbalanced dNTP pools, we reversed the orientation of the CAN1 gene (CAN1-OR2)

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Summary

Introduction

The integrity of an organism’s genome is vital to its continued survival, whether unicellular microbe or complex large mammal [1]. There are highly conserved mechanisms involved in regulating and protecting genetic material both during and post DNA replication. One of the first safety systems for DNA replication is the stringent control of dNTP synthesis by ribonucleotide reductase (RNR), which maintains concentrations of the individual dNTPs at different levels [1,2]. The large subunits contain allosteric specificity sites that modulate enzyme activity and control the balance of the four dNTPs by influencing the specific ribonucleoside diphosphate reduction reaction within the catalytic sites [5]. A highly conserved loop of 13 amino acid residues (Loop 2) connects the allosteric specificity and catalytic sites and is crucial for the correct allosteric regulation of the enzyme [6,7]

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