A DNA replication origin and a replication fork barrier used in vivo in the circular plasmid pKD1

Abstract.

As in other yeasts, ARS-containing plasmids can be maintained extrachromosomally in Kluyveromyces lactis. Although some fragments of K. lactis DNA have ARS activity in both K. lactis and Saccharomyces cerevisiae, it appears that the sequences required for ARS activity in the two yeasts are different. As an approach to a better understanding of ARS structure and function in K. lactis, we analyzed the replication of the circular plasmid pKD1. We identified a 159-bp sequence able to promote autonomous replication of pKD1 in both yeasts; this fragments contains both a sequence related to the S. cerevisiae ARS consensus sequence and a region of 53% identity to the 40-bp sequence essential for K. lactis KARS101 function. By the analysis of in vivo replication intermediates we provide the first direct evidence that DNA replication initiates at or near the K. lactis ARS element. Replication terminates at the cis-acting stability locus of pKD1, which functions as a replication fork barrier (RFB) and is necessary for proper plasmid segregation. RFB activity requires the pKD1 gene products that are important for plasmid segregation, suggesting a link between DNA replication termination and plasmid segregation in a eukaryotic organism.