Mechanism of illegitimate recombination in Kluyveromyces lactis.
Andreas Kegel, Sidney Carter, Stefan Äström
Developmental Biology, Stockholm University, Svante Arrheniusv 17, Stockholm, 10691, Sweden
Illegitimate recombination (IR) is the process by which two DNA sequences not sharing homology to each other are joined. IR can be measured by integrative transformation of cells using a linearized plasmid not sharing homology with the genome. In S. cerevisiae, IR is an inefficient process (1-5 colonies/µg plasmid DNA), but in Kluyveromyces lactis (milk yeast) IR occurred 1000-fold more frequently, indicating that IR mechanisms were different in the two yeasts. Using a plasmid rescue procedure we investigated IR in milk yeast, studying target site specificity and influence of topoisomerase I cleavage sites. In addition, using the complete genome sequence of milk yeast we investigated the global distribution of more than 100 integrations, which will be presented. We also explored the genetic requirements for both IR and homologous recombination in Kluyveromyces. IR was completely dependent on the NHEJ pathway for DSB-repair, suggesting that integrations by IR occurred at spontaneous mitotic DSBs. In contrast, ends-out integration of constructs sharing 500bp of homology to the genome was very inefficient, 99% of the transformants being targeted to nonhomologous loci. Strains mutationally compromised in the NHEJ pathway, however, exclusively performed gene targeting at the homologous locus. These observations were explored to generate a simple method for PCR-based gene targeting in milk yeast, and also a powerful mutagenesis method, similar to EP mutagenesis in Drosophila.
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