Escherichia coli DNA polymerase IV contributes to spontaneous mutagenesis at coding sequences but not microsatellite alleles

Kimberly D. Jacob, Kristin Eckert

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10 Citations (Scopus)

Abstract

Slipped strand mispairing during DNA synthesis is one proposed mechanism for microsatellite or short tandem repeat (STR) mutation. However, the DNA polymerase(s) responsible for STR mutagenesis have not been determined. In this study, we investigated the effect of the Escherichia coli dinB gene product (Pol IV) on mononucleotide and dinucleotide repeat stability, using an HSV-tk gene episomal reporter system for microsatellite mutations. For the control vector (HSV-tk gene only) we observed a statistically significant 3.5-fold lower median mutation frequency in dinB- than dinB+ cells (p < 0.001, Wilcoxon Mann Whitney Test). For vectors containing an in-frame mononucleotide allele ([G/C]10) or either of two dinucleotide alleles ([GT/CA]10 and [TC/AG]11) we observed no statistically significant difference in the overall HSV-tk mutation frequency observed between dinB+ and dinB- strains. To determine if a mutational bias exists for mutations made by Pol IV, mutational spectra were generated for each STR vector and strain. No statistically significant differences between strains were observed for either the proportion of mutational events at the STR or STR specificity among the three vectors. However, the specificity of mutational events at the STR alleles in each strain varied in a statistically significant manner as a consequence of microsatellite sequence. Our results indicate that while Pol IV contributes to spontaneous mutations within the HSV-tk coding sequence, Pol IV does not play a significant role in spontaneous mutagenesis at [G/C]10, [GT/CA]10, or [TC/AG]11 microsatellite alleles. Our data demonstrate that in a wild type genetic background, the major factor influencing microsatellite mutagenesis is the allelic sequence composition.

Original languageEnglish (US)
Pages (from-to)93-103
Number of pages11
JournalMutation Research - Fundamental and Molecular Mechanisms of Mutagenesis
Volume619
Issue number1-2
DOIs
StatePublished - Jun 1 2007

Fingerprint

DNA Polymerase beta
Mutagenesis
Microsatellite Repeats
Alleles
Escherichia coli
Mutation
Mutation Rate
pol Gene Products
Dinucleotide Repeats
DNA-Directed DNA Polymerase
Reporter Genes

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Genetics
  • Health, Toxicology and Mutagenesis

Cite this

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title = "Escherichia coli DNA polymerase IV contributes to spontaneous mutagenesis at coding sequences but not microsatellite alleles",
abstract = "Slipped strand mispairing during DNA synthesis is one proposed mechanism for microsatellite or short tandem repeat (STR) mutation. However, the DNA polymerase(s) responsible for STR mutagenesis have not been determined. In this study, we investigated the effect of the Escherichia coli dinB gene product (Pol IV) on mononucleotide and dinucleotide repeat stability, using an HSV-tk gene episomal reporter system for microsatellite mutations. For the control vector (HSV-tk gene only) we observed a statistically significant 3.5-fold lower median mutation frequency in dinB- than dinB+ cells (p < 0.001, Wilcoxon Mann Whitney Test). For vectors containing an in-frame mononucleotide allele ([G/C]10) or either of two dinucleotide alleles ([GT/CA]10 and [TC/AG]11) we observed no statistically significant difference in the overall HSV-tk mutation frequency observed between dinB+ and dinB- strains. To determine if a mutational bias exists for mutations made by Pol IV, mutational spectra were generated for each STR vector and strain. No statistically significant differences between strains were observed for either the proportion of mutational events at the STR or STR specificity among the three vectors. However, the specificity of mutational events at the STR alleles in each strain varied in a statistically significant manner as a consequence of microsatellite sequence. Our results indicate that while Pol IV contributes to spontaneous mutations within the HSV-tk coding sequence, Pol IV does not play a significant role in spontaneous mutagenesis at [G/C]10, [GT/CA]10, or [TC/AG]11 microsatellite alleles. Our data demonstrate that in a wild type genetic background, the major factor influencing microsatellite mutagenesis is the allelic sequence composition.",
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T1 - Escherichia coli DNA polymerase IV contributes to spontaneous mutagenesis at coding sequences but not microsatellite alleles

AU - Jacob, Kimberly D.

AU - Eckert, Kristin

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N2 - Slipped strand mispairing during DNA synthesis is one proposed mechanism for microsatellite or short tandem repeat (STR) mutation. However, the DNA polymerase(s) responsible for STR mutagenesis have not been determined. In this study, we investigated the effect of the Escherichia coli dinB gene product (Pol IV) on mononucleotide and dinucleotide repeat stability, using an HSV-tk gene episomal reporter system for microsatellite mutations. For the control vector (HSV-tk gene only) we observed a statistically significant 3.5-fold lower median mutation frequency in dinB- than dinB+ cells (p < 0.001, Wilcoxon Mann Whitney Test). For vectors containing an in-frame mononucleotide allele ([G/C]10) or either of two dinucleotide alleles ([GT/CA]10 and [TC/AG]11) we observed no statistically significant difference in the overall HSV-tk mutation frequency observed between dinB+ and dinB- strains. To determine if a mutational bias exists for mutations made by Pol IV, mutational spectra were generated for each STR vector and strain. No statistically significant differences between strains were observed for either the proportion of mutational events at the STR or STR specificity among the three vectors. However, the specificity of mutational events at the STR alleles in each strain varied in a statistically significant manner as a consequence of microsatellite sequence. Our results indicate that while Pol IV contributes to spontaneous mutations within the HSV-tk coding sequence, Pol IV does not play a significant role in spontaneous mutagenesis at [G/C]10, [GT/CA]10, or [TC/AG]11 microsatellite alleles. Our data demonstrate that in a wild type genetic background, the major factor influencing microsatellite mutagenesis is the allelic sequence composition.

AB - Slipped strand mispairing during DNA synthesis is one proposed mechanism for microsatellite or short tandem repeat (STR) mutation. However, the DNA polymerase(s) responsible for STR mutagenesis have not been determined. In this study, we investigated the effect of the Escherichia coli dinB gene product (Pol IV) on mononucleotide and dinucleotide repeat stability, using an HSV-tk gene episomal reporter system for microsatellite mutations. For the control vector (HSV-tk gene only) we observed a statistically significant 3.5-fold lower median mutation frequency in dinB- than dinB+ cells (p < 0.001, Wilcoxon Mann Whitney Test). For vectors containing an in-frame mononucleotide allele ([G/C]10) or either of two dinucleotide alleles ([GT/CA]10 and [TC/AG]11) we observed no statistically significant difference in the overall HSV-tk mutation frequency observed between dinB+ and dinB- strains. To determine if a mutational bias exists for mutations made by Pol IV, mutational spectra were generated for each STR vector and strain. No statistically significant differences between strains were observed for either the proportion of mutational events at the STR or STR specificity among the three vectors. However, the specificity of mutational events at the STR alleles in each strain varied in a statistically significant manner as a consequence of microsatellite sequence. Our results indicate that while Pol IV contributes to spontaneous mutations within the HSV-tk coding sequence, Pol IV does not play a significant role in spontaneous mutagenesis at [G/C]10, [GT/CA]10, or [TC/AG]11 microsatellite alleles. Our data demonstrate that in a wild type genetic background, the major factor influencing microsatellite mutagenesis is the allelic sequence composition.

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