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Development of a supF-based mutation-detection system in the extreme thermophile Thermus thermophilus HB27

  • Yoichiro Togawa
  • Shiori Shiotani
  • Yuki Kato
  • Kazune Ezaki
  • Tatsuo Nunoshiba
  • Keiichiro HiratsuEmail author
Methods Paper
  • 31 Downloads

Abstract

Thermus thermophilus (T. thermophilus) HB27 is an extreme thermophile that grows optimally at 65–72 °C. Heat-induced DNA lesions are expected to occur at a higher frequency in the genome of T. thermophilus than in those of mesophiles; however, the mechanisms underlying the maintenance of genome integrity at high temperatures remain poorly understood. The study of mutation spectra has become a powerful approach to understanding the molecular mechanisms responsible for DNA repair and mutagenesis in mesophilic species. Therefore, we developed a supF-based system to detect a broad spectrum of mutations in T. thermophilus. This system was validated by measuring spontaneous mutations in the wild type and a udgA, B double mutant deficient in uracil-DNA glycosylase (UDG) activity. We found that the mutation frequency of the udgA, B strain was 4.7-fold higher than that of the wild type and G:C→A:T transitions dominated, which was the most reasonable for the mutator phenotype associated with the loss of UDG function in T. thermophilus. These results show that this system allowed for the rapid analysis of mutations in T. thermophilus, and may be useful for studying the molecular mechanisms responsible for DNA repair and mutagenesis in this extreme thermophile.

Keywords

Thermus thermophilus supF Mutation Shuttle vector Uracil-DNA glycosylase DNA repair 

Notes

Acknowledgements

We thank Prof. Isao Kuraoka (Fukuoka University) for helpful discussion. This work was supported by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

438_2019_1565_MOESM1_ESM.pdf (469 kb)
Supplementary material 1 (PDF 470 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Applied ChemistryNational Defense AcademyYokosukaJapan
  2. 2.College of Liberal ArtsInternational Christian UniversityMitakaJapan

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