I-SceI endonuclease: a new tool for DNA repair studies and genetic manipulations in streptomycetes |
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Authors: | Theresa Siegl Lutz Petzke Elisabeth Welle Andriy Luzhetskyy |
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Institution: | (1) Department of Pharmaceutical Biology and Biotechnology, Albert-Ludwigs-University of Freiburg, Stefan-Meier-st. 19, Freiburg, 79104, Germany; |
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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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