The fidelity of DNA replication, particularly on GC-rich templates, is reduced by defects of the Fe-S cluster in DNA polymerase δ.

Denis A Kiktev,Thomas A Kunkel,Elena I Stepchenkova,Adam Burkholder,Peter M Burgers,Piotr Mieczkowski,Joseph Dahl,Tony Zhang,Margaret Dominska,Scott Lujan,Thomas D Petes

doi:10.1093/nar/gkab371

Abstract

Iron-sulfur clusters (4Fe–4S) exist in many enzymes concerned with DNA replication and repair. The contribution of these clusters to enzymatic activity is not fully understood. We identified the MET18 (MMS19) gene of Saccharomyces cerevisiae as a strong mutator on GC-rich genes. Met18p is required for the efficient insertion of iron-sulfur clusters into various proteins. met18 mutants have an elevated rate of deletions between short flanking repeats, consistent with increased DNA polymerase slippage. This phenotype is very similar to that observed in mutants of POL3 (encoding the catalytic subunit of Pol δ) that weaken binding of the iron-sulfur cluster. Comparable mutants of POL2 (Pol ϵ) do not elevate deletions. Further support for the conclusion that met18 strains result in impaired DNA synthesis by Pol δ are the observations that Pol δ isolated from met18 strains has less bound iron and is less processive in vitro than the wild-type holoenzyme.

Highlights

Mutations must occur frequently enough to generate variants suited for evolution, but infrequently enough that essential genes are not often inactivated
We found that the high mutation rate of the URA3-GC allele was a consequence of a strongly elevated rate of deletions between direct repeats as well as an elevated rate of single-base mutations caused by increased recruitment of the error-prone Pol ␨
The variant form of Pol ␦ that lacks the Fe–S cluster or has a destabilized Fe–S cluster has an elevated rate of DNA polymerase slippage/template switching in vivo and decreased processivity in vitro

Summary

Introduction

Mutations must occur frequently enough to generate variants suited for evolution, but infrequently enough that essential genes are not often inactivated. 7), the rate of single-base mutations is about 3 × 10−10/bp/division, and the rate of small insertion/deletions (in/dels) is at least 10-fold lower. The relatively low in vivo mutation rate for single-base mutations in eukaryotes is a consequence of three processes that operate sequentially: accurate base selectivity of the replicative polymerases (Pols) ␣, ␦ and ε; a proofreading exonuclease associated with Pols ␦ and ε, and the error-correcting mismatch repair (MMR) system [8]. The catalytic subunits of DNA Pol ␦ and ε associate with other proteins (such as PCNA) that increase their processivity. These associations reduce the frequency of insertion/deletions (in/dels), mutations that likely reflect DNA polymerase slippage [9]. Some issues remain controversial, in unstressed wild-type cells, synthesis on the leading and lagging strands is primarily performed by Pols ε and ␦, respectively; in certain mutant backgrounds and in certain genomic regions in wild-type cells, Pol ␦ replicates both leading and lagging strands [10,11]

Methods

Results

Conclusion