An SSB encoded by and operating on linear killer plasmids from Kluyveromyces lactis.

Abstract

The Kluyveromyces lactis linear plasmids k1 and k2 belong to the family of protein-primed linear DNA genomes, which includes adenoviruses. Here we identify the 18 kDa gene product of k2ORF5 as a novel putative single-stranded DNA binding protein, SSB. As judged from Western analysis using an epitope-tagged fusion protein and ssDNA-agarose affinity chromatography, the Orf5 protein preferentially binds to ssDNA in vitro. Consistently, electrophoretic mobility shift assays demonstrate that ssDNA plasmid probes from k1 and k2 are retarded by this Orf5-associated SSB activity. ORF5 gene shuffle-mediated mutagenesis in vivo results in k1/k2 plasmid instability, pointing towards a role for the Orf5 protein in plasmid replication. Consistently, the Orf5 protein protects ssDNA from exonuclease digestion and stimulates Klenow enzyme. Our findings suggest a functional role for the Orf5 protein as a putative SSB probably required during k1/k2 plasmid DNA synthesis.