Relocation of a cytoplasmic yeast linear plasmid to the nucleus is associated with circularization via nonhomologous recombination involving invertedterminal repeats

The linear plasmid pCLU1 from the yeast Kluyveromyces lactis normally repli cates in the cytoplasm, with the aid of the helper linear plasmid pGKL2, us ing terminal protein (TP) as a primer. However, it relocates to the nucleus when selection is applied for the expression of a plasmid-borne nuclear ma rker. Migration to the nucleus occurred in K. lactis at a frequency of abou t 10(-3)/cell ten or more times higher than the rate observed in Saccharomy ces cerevisiae. The nuclear plasmids existed only in a circularized form in K. lactis, while in S, cerevisiae a telomere-associated linear form is als o found. Sequence analysis showed that circularization in K. lactis was cau sed by non-homologous recombination between the inverted terminal repeat (I TR) at the ends of the linear form and non-specific internal target sites i n pCLU1. No sequence similarity existed among the junction sites, indicatin g that the free ITR end plays a crucial role in circularization. In S. cere visiae, circular plasmids were generated not only by nonhomologous recombin ation, but also by homologous recombination between short direct repeats wi thin pCLU1. Circularization via the ITR end was observed independently of R AD52 activity. Sequences highly homologous to ARS core elements, 5'-ATTTATT GTTTT-3' for K. lactis and 5'- (A/T)TTTAT(T/G)TTT(A/T)-3' for S. cerevisiae , were detected at multiple sites in the nuclear forms of the plasmids.