The Kluyveromyces linear plasmids, pGKL1 and pGKL2, carrying terminal protein (TP), are located in the cytoplasm and have a unique gene expression system with the plasmid-specific promoter element termed UCS, which functions only in the cytoplasm. In this study we have developed an in vivo assay system in Saccharomyces cerevisiae which enables the detection of a rare migration of the yeast cytoplasmic plasmid to the nucleus, using a pGKL1-derived cytoplasmic linear plasmid pCLU1. pCLU1 had both the UCS-fused LEU2 gene (a cytoplasmic marker) and the native URA3 gene (a nuclear marker) and therefore its cytoplasmic-nucleo localized could be determined by the phenotypic analysis of the marker. The nuclearly migrated plasmids were often detected as linear plasmids having the telomere sequence of the host yeast at both ends, although circular plasmids were also found. The circular form was produced by the the terminal fusion of pCLU1. Insertion of a Ty element into a nuclearly migrated plasmid was observed, allowing the ROAM-regulated expression of the adjacent nuclearly silent UCS-fused LEU2 gene. The nuclearly located plasmids, whether linear or circular, were less sensitive to UV-mediated curing than pGKL and pCLU1.

The Kluyveromyces linear plasmids pGKL1 and pGKL2, encoding killer activity, were efficiently cured by UV irradiation. This event was investigated in more detail by the use of the terminal protein (TP)-associated cytoplasmic linear plasmids, pJKL1 and pRKL2, with a selectable marker LEU2. This observation was compared with the UV effect on the nuclear plasmids pLS1 (telomere-associated linear form) and YCp121 (centromere-integrated circular form), indicating that the UV hypersensitivity was specific to the cytoplasmic plasmids. Using rad4 and wild-type strains of S. cerevisiae, both pJKL1 and the nuclear plasmids were found to respond not only to photoreactivation repair but also to excision repair of UV-induced DNA damage. Thus these DNA repair systems were functional for both the nuclear and cytoplasmic plasmids in yeast, and it was suggested that the UV hypersensitivity of cytoplasmic plasmids might have been caused by a defect in other repair systems or in the TP-primed replication. Possibly TP-associated Debaryomyces linear plasmids were also UV hypersensitive.