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

Abstract

The linear plasmid pCLU1 from the yeast Kluyveromyces lactis normally replicates in the cytoplasm, with the aid of the helper linear plasmid pGKL2, using terminal protein (TP) as a primer. However, it relocates to the nucleus when selection is applied for the expression of a plasmid-borne nuclear marker. Migration to the nucleus occurred in K. lactis at a frequency of about 10(-3)/cell ten or more times higher than the rate observed in Saccharomyces cerevisiae. The nuclear plasmids existed only in a circularized form in K. lactis, while in S. cerevisiae a telomere-associated linear form is also found. Sequence analysis showed that circularization in K. lactis was caused by non-homologous recombination between the inverted terminal repeat (ITR) at the ends of the linear form and non-specific internal target sites in pCLU1. No sequence similarity existed among the junction sites, indicating that the free ITR end plays a crucial role in circularization. In S. cerevisiae, circular plasmids were generated not only by nonhomologous recombination, but also by homologous recombination between short direct repeats within pCLU1. Circularization via the ITR end was observed independently of RAD52 activity. Sequences highly homologous to ARS core elements, 5'-ATTTATTGTTTT-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.

Progressive rearrangement of telomeric sequences added to both the ITR ends of the yeast linear pGKL plasmid

“…The nuclearly relocated pCLU1 replicated in either circular or linear form. While the circular form resulted from pCLU1 by ITR-involved intramolecular recombination or transposon-like invasion (18), the linear plasmid pTLU was created by an addition of the host telomeric repeats at both ends of pCLU1 (15, 16). …”

“…pCLU1 carried terminal-protein (TP) at both ends and were normally maintained in the cytoplasm with the helper pGKL2. When relocated into the nucleus, however, pCLU1 replicated in a circular form (termed pRLU) or telomere-associated linear form (termed pTLU) independent of pGKL2 (18). …”