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I-SceI endonuclease: a new tool for DNA repair studies and genetic manipulations in streptomycetes

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Abstract

Actinomycetes are Gram-positive bacteria with a complex life cycle. They produce many pharmaceutically relevant secondary metabolites, including antibiotics and anticancer drugs. However, there is a limited number of biotechnological applications available as opposed to genetic model organisms like Bacillus subtilis or Escherichia coli. We report here a system for the functional expression of a synthetic gene encoding the I-SceI homing endonuclease in several streptomycetes. Using the synthetic sce(a) gene, we were able to create controlled genomic DNA double-strand breaks. A mutagenesis system, based on the homing endonuclease I-SceI, has been developed to construct targeted, non-polar, unmarked gene mutations in Streptomyces sp. Tü6071. In addition, we have shown that homologous recombination is a major pathway in streptomycetes to repair an I-SceI-generated DNA double-strand break. This novel I-SceI-based tool will be useful in fundamental studies on the repair mechanism of DNA double-strand breaks and for a variety of biotechnological applications.

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Acknowledgment

We are very grateful to Prof. A. Bechthold for critical discussions during the work as well as for the financial support. The authors also wish to thank Dr. B. Gust for providing plasmids pIJ778 and pIJ773, as well as Dr. M. Daum and K. Dietmann for the help in plasmid construction.

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Authors and Affiliations

  1. Department of Pharmaceutical Biology and Biotechnology, Albert-Ludwigs-University of Freiburg, Stefan-Meier-st. 19, Freiburg, 79104, Germany

    Theresa Siegl, Lutz Petzke, Elisabeth Welle & Andriy Luzhetskyy

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  1. Theresa Siegl
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  2. Lutz Petzke
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  3. Elisabeth Welle
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  4. Andriy Luzhetskyy
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Correspondence to Andriy Luzhetskyy.

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Siegl, T., Petzke, L., Welle, E. et al. I-SceI endonuclease: a new tool for DNA repair studies and genetic manipulations in streptomycetes. Appl Microbiol Biotechnol 87, 1525–1532 (2010). https://doi.org/10.1007/s00253-010-2643-y

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  • DOI: https://doi.org/10.1007/s00253-010-2643-y

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