%0 Journal Article
%T Whole-Genome Profiling of a Novel Mutagenesis Technique Using Proofreading-Deficient DNA Polymerase
%A Yuh Shiwa
%A Sanae Fukushima-Tanaka
%A Ken Kasahara
%A Takayuki Horiuchi
%A Hirofumi Yoshikawa
%J International Journal of Evolutionary Biology
%D 2012
%I Hindawi Publishing Corporation
%R 10.1155/2012/860797
%X A novel mutagenesis technique using error-prone DNA polymerase ¦Ä (pol¦Ä), the disparity mutagenesis model of evolution, has been successfully employed to generate novel microorganism strains with desired traits. However, little else is known about the spectra of mutagenic effects caused by disparity mutagenesis. We evaluated and compared the performance of the pol¦ÄMKII mutator, which expresses the proofreading-deficient and low-fidelity pol¦Ä, in Saccharomyces cerevisiae haploid strain with that of the commonly used chemical mutagen ethyl methanesulfonate (EMS). This mutator strain possesses exogenous mutant pol¦Ä supplied from a plasmid, tthereby leaving the genomic one intact. We measured the mutation rate achieved by each mutagen and performed high-throughput next generation sequencing to analyze the genome-wide mutation spectra produced by the 2 mutagenesis methods. The mutation frequency of the mutator was approximately 7 times higher than that of EMS. Our analysis confirmed the strong G/C to A/T transition bias of EMS, whereas we found that the mutator mainly produces transversions, giving rise to more diverse amino acid substitution patterns. Our present study demonstrated that the pol¦ÄMKII mutator is a useful and efficient method for rapid strain improvement based on in vivo mutagenesis. 1. Introduction Random mutagenesis is a powerful tool for generating enzymes, proteins, metabolic pathways, or even entire genomes with desired or improved properties [1]. Due to the technical simplicity and applicability to almost any organism, chemical or radiation mutagenesis is frequently used for the generation of genetic variability in a microorganism. However, these methods tend to be inefficient because they can cause substantial cell damage when performed in vivo [2]. A novel mutagenesis technique using error-prone DNA polymerase ¦Ä (pol¦Ä), based on the disparity mutagenesis model of evolution [3] has been successfully employed to generate novel microorganism strains with desired traits [4¨C11]. In the disparity model, mutations occur preferentially on the lagging strand, due to the more complex, discontinuous DNA replication that takes place there. Computer simulation shows that the disparity model accumulates more mutations than the parity model, in which mutations occur stochastically and evenly in both strands [3]. In addition, the disparity model produces greater diversity because some offspring will have mutant DNA while some offspring will have nonmutated, wild-type DNA. Several studies have shown that the disparity mutagenesis method often achieved
%U http://www.hindawi.com/journals/ijeb/2012/860797/