Nonhomologous end joining and homologous recombination DNA repair pathways in integration mutagenesis in the xylose-fermenting yeastPichia stipitis
| Autor: | Stefan Freese, Ulrich Klinner, Nicole Maassen, Volkmar Passoth, Barbara Schruff |
|---|---|
| Rok vydání: | 2008 |
| Předmět: |
Recombination
Genetic Genetics DNA Repair biology Mutagenesis General Medicine biology.organism_classification Applied Microbiology and Biotechnology Microbiology Pichia Homologous Recombination Pathway Non-homologous end joining Mutagenesis Insertional Transformation (genetics) chemistry.chemical_compound Transformation Genetic chemistry Cotransformation DNA Fungal Pichia stipitis DNA Sequence Deletion Transformation efficiency |
| Zdroj: | FEMS Yeast Research. 8:735-743 |
| ISSN: | 1567-1364 1567-1356 |
| DOI: | 10.1111/j.1567-1364.2008.00383.x |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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