title: Enhanced targeted integration mediated by translocated I-SceI during the Agrobacterium mediated transformation of yeast.
abstract: Agrobacterium mediated transformation (AMT) has been embraced by biotechnologists as the technology of choice to introduce or alter genetic traits of plants. However, in plants it is virtually impossible to predetermine the integration site of the transferred T-strand unless one is able to generate a double stranded break (DSB) in the DNA at the site of interest. In this study, we used the model organism Saccharomyces cerevisiae to investigate whether the Agrobacterium mediated translocation of site-specific endonucleases via the type IV secretion system (T4SS), concomitantly with T-DNA transfer is possible and whether this can improve the gene targeting efficiency. In addition to that, the effect of different chromatin states on targeted integration, was investigated. It was found that Agrobacterium mediated translocation of the homing endonuclease I-SceI has a positive effect on the integration of T-DNA via the homologous repair (HR) pathway. Furthermore, we obtained evidence that nucleosome removal has a positive effect on I-SceI facilitated T-DNA integration by HR. Reversely; inducing nucleosome formation at the site of integration removes the positive effect of translocated I-SceI on T-DNA integration.
authors: Department of Molecular and Developmental Genetics, Institute of Biology Leiden, Leiden, Sylviusweg 72, 2333 BE, The Netherlands.
authors: Martijn
authors: M
authors: Rolloos
authors: Department of Molecular and Developmental Genetics, Institute of Biology Leiden, Leiden, Sylviusweg 72, 2333 BE, The Netherlands.
authors: Paul J J
authors: PJ
authors: Hooykaas
authors: Department of Molecular and Developmental Genetics, Institute of Biology Leiden, Leiden, Sylviusweg 72, 2333 BE, The Netherlands.
authors: Bert J
authors: BJ
authors: van der Zaal
mesh_terms: D060054:Agrobacterium / Q000235:genetics* / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D014170:Transformation, Genetic
chemical_list: D029701:Saccharomyces cerevisiae Proteins; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific