Misalignment-mediated DNA polymerase β mutations: Comparison of microsatellite and frame-shift error rates using a forward mutation assay

Kristin Eckert, Andrew Mowery, Suzanne E. Hile

Research output: Contribution to journalArticle

50 Citations (Scopus)

Abstract

Mutations arising in microsatellite DNA are associated with neurological diseases and cancer. To elucidate the molecular basis of microsatellite mutation, we have determined the in vitro polymerase error frequencies at microsatellite sequences representative of those found in the human genome: [GT/CA]10, [TC/AG]11, and [TTCC/AAGG]9. DNA templates contained the microsatellites inserted in-frame into the 5′ region of the herpes simplex virus thymidine kinase (HSV-tk) gene. Polymerase β (polβ) error frequencies were quantitated in microsatellite sequences, relative to frame-shift error frequencies in coding sequences, from the same DNA synthesis reaction. The polβ error frequencies within the dinucleotide sequences were (2-9) × 10-3, 14-72-fold higher than the ssDNA template frequencies. The polβ error frequencies within the tetranucleotide sequences were (4-6) × 10-3, a 4-13-fold increase over background. Strand biases were observed for the [TC/AG]11 and [TTCC/AAGG]9 alleles, in which more errors were produced when the purine strand served as a template. Mutations within each microsatellite included noncanonical base substitution events and single nucleotide deletions as well as the expected unit length changes. An exponential relationship was observed between the polymerase error frequency per site and both the number of repetitive units and total length of the allele. Our observations are consistent with the strand slippage model of microsatellite mutagenesis and demonstrate that DNA sequence and/or structural differences result in mutational strand biases. To our knowledge, this is the first direct quantitation of DNA polymerase errors in vitro using template microsatellite sequences.

Original languageEnglish (US)
Pages (from-to)10490-10498
Number of pages9
JournalBiochemistry
Volume41
Issue number33
DOIs
StatePublished - Aug 20 2002

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DNA-Directed DNA Polymerase
Microsatellite Repeats
Assays
Mutation
DNA
Genes
Alleles
Mutagenesis
Thymidine Kinase
DNA sequences
Human Genome
Simplexvirus
Viruses
Substitution reactions
Nucleotides

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

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abstract = "Mutations arising in microsatellite DNA are associated with neurological diseases and cancer. To elucidate the molecular basis of microsatellite mutation, we have determined the in vitro polymerase error frequencies at microsatellite sequences representative of those found in the human genome: [GT/CA]10, [TC/AG]11, and [TTCC/AAGG]9. DNA templates contained the microsatellites inserted in-frame into the 5′ region of the herpes simplex virus thymidine kinase (HSV-tk) gene. Polymerase β (polβ) error frequencies were quantitated in microsatellite sequences, relative to frame-shift error frequencies in coding sequences, from the same DNA synthesis reaction. The polβ error frequencies within the dinucleotide sequences were (2-9) × 10-3, 14-72-fold higher than the ssDNA template frequencies. The polβ error frequencies within the tetranucleotide sequences were (4-6) × 10-3, a 4-13-fold increase over background. Strand biases were observed for the [TC/AG]11 and [TTCC/AAGG]9 alleles, in which more errors were produced when the purine strand served as a template. Mutations within each microsatellite included noncanonical base substitution events and single nucleotide deletions as well as the expected unit length changes. An exponential relationship was observed between the polymerase error frequency per site and both the number of repetitive units and total length of the allele. Our observations are consistent with the strand slippage model of microsatellite mutagenesis and demonstrate that DNA sequence and/or structural differences result in mutational strand biases. To our knowledge, this is the first direct quantitation of DNA polymerase errors in vitro using template microsatellite sequences.",
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Misalignment-mediated DNA polymerase β mutations : Comparison of microsatellite and frame-shift error rates using a forward mutation assay. / Eckert, Kristin; Mowery, Andrew; Hile, Suzanne E.

In: Biochemistry, Vol. 41, No. 33, 20.08.2002, p. 10490-10498.

Research output: Contribution to journalArticle

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T2 - Comparison of microsatellite and frame-shift error rates using a forward mutation assay

AU - Eckert, Kristin

AU - Mowery, Andrew

AU - Hile, Suzanne E.

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AB - Mutations arising in microsatellite DNA are associated with neurological diseases and cancer. To elucidate the molecular basis of microsatellite mutation, we have determined the in vitro polymerase error frequencies at microsatellite sequences representative of those found in the human genome: [GT/CA]10, [TC/AG]11, and [TTCC/AAGG]9. DNA templates contained the microsatellites inserted in-frame into the 5′ region of the herpes simplex virus thymidine kinase (HSV-tk) gene. Polymerase β (polβ) error frequencies were quantitated in microsatellite sequences, relative to frame-shift error frequencies in coding sequences, from the same DNA synthesis reaction. The polβ error frequencies within the dinucleotide sequences were (2-9) × 10-3, 14-72-fold higher than the ssDNA template frequencies. The polβ error frequencies within the tetranucleotide sequences were (4-6) × 10-3, a 4-13-fold increase over background. Strand biases were observed for the [TC/AG]11 and [TTCC/AAGG]9 alleles, in which more errors were produced when the purine strand served as a template. Mutations within each microsatellite included noncanonical base substitution events and single nucleotide deletions as well as the expected unit length changes. An exponential relationship was observed between the polymerase error frequency per site and both the number of repetitive units and total length of the allele. Our observations are consistent with the strand slippage model of microsatellite mutagenesis and demonstrate that DNA sequence and/or structural differences result in mutational strand biases. To our knowledge, this is the first direct quantitation of DNA polymerase errors in vitro using template microsatellite sequences.

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