engleski

Plasmid integration is preferred in the C-terminal part of the yeast ura3 gene

Double-strand breaks in DNA (DSBs) are potent initiators of homologous recombination. In meiotic yeast cells, induction of DSBs is genetically controlled process. The breaks are not randomly distributed and are preferentially located in promotor regions. In mitotic cells, initiation of genetic recombination can be targeted to a specific region by restriction enzyme-induced DSB in transforming plasmid molecule. Reciprocal exchange between homologous sequences involved in recombination will result in integration of the plasmid molecule into yeast genome. However, the precise site of resolution of the recombinational intermediate can not be determined if the recombining sequences are completely homologous. In our work we studied DSB-induced recombination between plasmid URA3 gene and chromosomal ura3 allele interrupted by a heterologous insertion ( 6.1 kb Ty element or 2.1 kb ARG4 gene). Circular double-stranded (but not single-stranded!) plasmids were mainly integrated to the "right" side from insertion (44/48 integrants) although two target homologies were of comparable size (541 on the left and 630 bps on the right). When different restriction enzymes were used to direct recombination in homologous sequence on either side of the insert we found out that some recombination events initiated on one side of the long heterologous insert were resolved on the other side. However, such displaced events were detected almost exclusively when integrations were targeted to the "left" from insertion (N-terminal part of the gene). The frequency of targeted integrations was also dependent on the type and orientation of the insert and on the proximity of the DSB to the insert. These results suggest that (i) homologous recombination between plasmid molecule and yeast chromosome can involve long heterologous regions and (ii) that recombination events are preferentially resolved in the C-terminal part of the ura3 gene. We also present a molecular model for homologous recombination involving long heterologous regions.

homologous recombination; plasmid integration; Saccharomyces cerevisiae

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