I-SceI endonuclease: a new tool for DNA repair studies and genetic manipulations in streptomyces.
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.
Siegl
Theresa
T
Department of Pharmaceutical Biology and Biotechnology, Albert-Ludwigs-University of Freiburg, Stefan-Meier-st. 19, Freiburg 79104, Germany.
Petzke
Lutz
L
Welle
Elisabeth
E
Luzhetskyy
Andriy
A
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D003850:Deoxyribonuclease I / Q000235:genetics / Q000378:metabolism*; D005813:Genes, Synthetic; D005821:Genetic Techniques; D016296:Mutagenesis; D011995:Recombination, Genetic; D013302:Streptomyces / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D003850:Deoxyribonuclease I