A novel mutagenesis technique using error-prone DNA polymerase δ (polδ), 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 polδMKII mutator, which expresses the proofreading-deficient and low-fidelity polδ, in Saccharomyces cerevisiae haploid strain with that of the commonly used chemical mutagen ethyl methanesulfonate (EMS). This mutator strain possesses exogenous mutant polδ 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 polδMKII 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 δ (polδ), based on the disparity mutagenesis model of evolution [3] has been successfully employed to generate novel microorganism strains with desired traits [4–11]. 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
References
[1]
N. E. Labrou, “Random mutagenesis methods for in vitro directed enzyme evolution,” Current Protein and Peptide Science, vol. 11, no. 1, pp. 91–100, 2010.
[2]
O. Selifonova, F. Valle, and V. Schellenberger, “Rapid evolution of novel traits in microorganisms,” Applied and Environmental Microbiology, vol. 67, no. 8, pp. 3645–3649, 2001.
[3]
M. Furusawa and H. Doi, “Promotion of evolution: disparity in the frequency of strand-specific misreading between the lagging and leading DNA strands enhances disproportionate accumulation of mutations,” Journal of Theoretical Biology, vol. 157, no. 1, pp. 127–133, 1992.
[4]
K. Tanabe, T. Kondo, Y. Onodera, and M. Furusawa, “A conspicuous adaptability to antibiotics in the Escherichia coli mutator strain, dnaQ49,” FEMS Microbiology Letters, vol. 176, no. 1, pp. 191–196, 1999.
[5]
C. Shimoda, A. Itadani, A. Sugino, and M. Furusawa, “Isolation of thermotolerant mutants by using proofreading-deficient DNA polymerase δ as an effective mutator in Saccharomyces cerevisiae,” Genes and Genetic Systems, vol. 81, no. 6, pp. 391–397, 2006.
[6]
M. Itakura, K. Tabata, S. Eda et al., “Generation of Bradyrhizobium japonicum mutants with increased N2O reductase activity by selection after introduction of a mutated dnaQ gene,” Applied and Environmental Microbiology, vol. 74, no. 23, pp. 7258–7264, 2008.
[7]
H. Abe, Y. Fujita, Y. Chiba, Y. Jigami, and K. I. Nakayama, “Upregulation of genes involved in gluconeogenesis and the glyoxylate cycle suppressed the drug sensitivity of an N-glycan-deficient Saccharomyces cerevisiae mutant,” Bioscience, Biotechnology and Biochemistry, vol. 73, no. 6, pp. 1398–1403, 2009.
[8]
H. Abe, Y. Fujita, Y. Takaoka et al., “Ethanol-tolerant Saccharomyces cerevisiae strains isolated under selective conditions by over-expression of a proofreading-deficient DNA polymerase δ,” Journal of Bioscience and Bioengineering, vol. 108, no. 3, pp. 199–204, 2009.
[9]
H. Abe, Y. Takaoka, Y. Chiba et al., “Development of valuable yeast strains using a novel mutagenesis technique for the effective production of therapeutic glycoproteins,” Glycobiology, vol. 19, no. 4, pp. 428–436, 2009.
[10]
E. Y. Park, Y. Ito, M. Nariyama, et al., “The improvement of riboflavin production in Ashbya gossypii via disparity mutagenesis and DNA microarray analysis,” Applied Microbiology and Biotechnology, vol. 91, no. 5, pp. 1315–1326, 2011.
[11]
T. Matsuzawa, Y. Fujita, N. Tanaka, H. Tohda, A. Itadani, and K. Takegawa, “New insights into galactose metabolism by Schizosaccharomyces pombe: isolation and characterization of a galactose-assimilating mutant,” Journal of Bioscience and Bioengineering, vol. 111, no. 2, pp. 158–166, 2011.
[12]
S. Flibotte, M. L. Edgley, I. Chaudhry et al., “Whole-genome profiling of mutagenesis in Caenorhabditis elegans,” Genetics, vol. 185, no. 2, pp. 431–441, 2010.
[13]
T. Fukui, K. Yamauchi, T. Muroya et al., “Distinct roles of DNA polymerases delta and epsilon at the replication fork in Xenopus egg extracts,” Genes to Cells, vol. 9, no. 3, pp. 179–191, 2004.
[14]
L. Li, K. M. Murphy, U. Kanevets, and L. J. Reha-Krantz, “Sensitivity to phosphonoacetic acid: a new phenotype to probe DNA polymerase δ in Saccharomyces cerevisiae,” Genetics, vol. 170, no. 2, pp. 569–580, 2005.
[15]
A. Morrison, A. L. Johnson, L. H. Johnston, and A. Sugino, “Pathway correcting DNA replication errors in Saccharomyces cerevisiae,” The EMBO Journal, vol. 12, no. 4, pp. 1467–1473, 1993.
[16]
A. Morrison and A. Sugino, “The 3′→5′exonucleases of both DNA polymerases δ and ε participate in correcting errors of DNA replication in Saccharomyces cerevisiae,” Molecular and General Genetics, vol. 242, no. 3, pp. 289–296, 1994.
[17]
S. A. N. McElhinny, C. M. Stith, P. M. J. Burgers, and T. A. Kunkel, “Inefficient proofreading and biased error rates during inaccurate DNA synthesis by a mutant derivative of Saccharomyces cerevisiae DNA polymerase,” The Journal of Biological Chemistry, vol. 282, no. 4, pp. 2324–2332, 2007.
[18]
R. N. Venkatesan, J. J. Hsu, N. A. Lawrence, B. D. Preston, and L. A. Loeb, “Mutator phenotypes caused by substitution at a conserved motif A residue in eukaryotic DNA polymerase δ,” The Journal of Biological Chemistry, vol. 281, no. 7, pp. 4486–4494, 2006.
[19]
B. Timmermann, S. Jarolim, H. Russmayer et al., “A new dominant peroxiredoxin allele identified by whole-genome re-sequencing of random mutagenized yeast causes oxidant-resistance and premature aging,” Aging, vol. 2, no. 8, pp. 475–486, 2010.
[20]
R. D. Gietz and A. Sugino, “New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites,” Gene, vol. 74, no. 2, pp. 527–534, 1988.
[21]
D. E. Lea and C. A. Coulson, “The distribution of the numbers of mutants in bacterial populations,” Journal of Genetics, vol. 49, no. 3, pp. 264–285, 1949.
[22]
H. Li and R. Durbin, “Fast and accurate short read alignment with Burrows-Wheeler transform,” Bioinformatics, vol. 25, no. 14, pp. 1754–1760, 2009.
[23]
H. Li, B. Handsaker, A. Wysoker et al., “The sequence alignment/map format and SAMtools,” Bioinformatics, vol. 25, no. 16, pp. 2078–2079, 2009.
[24]
J. T. Robinson, H. Thorvaldsdóttir, W. Winckler et al., “Integrative genomics viewer,” Nature Biotechnology, vol. 29, no. 1, pp. 24–26, 2011.
[25]
S. Henikoff and J. G. Henikoff, “Amino acid substitution matrices from protein blocks,” Proceedings of the National Academy of Sciences of the United States of America, vol. 89, no. 22, pp. 10915–10919, 1992.
[26]
M. Lynch, W. Sung, K. Morris et al., “A genome-wide view of the spectrum of spontaneous mutations in yeast,” Proceedings of the National Academy of Sciences of the United States of America, vol. 105, no. 27, pp. 9272–9277, 2008.
[27]
T. S. Wong, D. Roccatano, M. Zacharias, and U. Schwaneberg, “A statistical analysis of random mutagenesis methods used for directed protein evolution,” Journal of Molecular Biology, vol. 355, no. 4, pp. 858–871, 2006.
[28]
T. S. Rasila, M. I. Pajunen, and H. Savilahti, “Critical evaluation of random mutagenesis by error-prone polymerase chain reaction protocols, Escherichia coli mutator strain, and hydroxylamine treatment,” Analytical Biochemistry, vol. 388, no. 1, pp. 71–80, 2009.
[29]
M. Furusawa, “Implications of double-stranded DNA structure for development, cancer and evolution,” Open Journal of Genetics, vol. 01, no. 03, pp. 78–87, 2011.
