title: Nonhomologous end joining and homologous recombination DNA repair pathways in integration mutagenesis in the xylose-fermenting yeast Pichia stipitis.
abstract: Pichia stipitis integrates linear homologous DNA fragments mainly ectopically. High rates of randomly occurring integration allow tagging mutagenesis with high efficiency using simply PCR amplificates of suitable selection markers from the P. stipitis genome. Linearization of an autonomously replicating vector caused a distinct increase of the transformation efficiency compared with the circular molecule. Cotransformation of a restriction endonuclease further enhanced the transformation efficiency. This effect was also observed with integrative vector DNA. In most cases vector integration in chromosomal targets did not depend on microhomologies, indicating that restriction-enzyme-mediated integration (REMI) does not play an essential role in P. stipitis. Small deletions were observed at the ends of the integrated vectors and in the target sites. Disruption of the PsKU80 gene increased the frequency of homologous integration considerably but resulted in a remarkable decrease of the transformation efficiency. These results suggest that in P. stipitis the nonhomologous end joining (NHEJ) pathway obviously predominates the homologous recombination pathway of double-strand break repair.
authors.affiliation: Department of Biology IV (Microbiology und Genetics), Unit Applied Microbiology, RWTH Aachen University, Germany.
authors.forename: Nicole
authors.initials: N
authors.lastname: Maassen
authors.forename: Stefan
authors.initials: S
authors.lastname: Freese
authors.forename: Barbara
authors.initials: B
authors.lastname: Schruff
authors.forename: Volkmar
authors.initials: V
authors.lastname: Passoth
authors.forename: Ulrich
authors.initials: U
authors.lastname: Klinner
mesh_terms: D004260:DNA Repair; D004271:DNA, Fungal / Q000235:genetics; D016254:Mutagenesis, Insertional; D010843:Pichia / Q000502:physiology*; D011995:Recombination, Genetic; D017384:Sequence Deletion; D014170:Transformation, Genetic
chemical_list: D004271:DNA, Fungal