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

As in other yeasts, ARS-containing plasmids can be maintained extrachromoso mally in Kluyveromyces lactis. Although some fragments of K. lactis DNA hav e ARS activity in both K. lactis and Saccharomyces cerevisiae, it appears t hat 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 id entified a 159-bp sequence able to promote autonomous replication of pKD1 i n both yeasts; this fragments contains both a sequence related to the S. ce revisiae ARS consensus sequence and a region of 53% identity to the 40-bp s equence essential for K. lactis KARS101 function. By the analysis of in viv o replication intermediates we provide the first direct evidence that DNA r eplication initiates at or near the K. lactis ARS element. Replication term inates at the cis-acting stability locus of pKD1, which functions as a repl ication fork barrier (RFB) and is necessary for proper plasmid segregation. RFB activity requires the pKD1 gene products that are important for plasmi d segregation, suggesting a link between DNA replication termination and pl asmid segregation in a eukaryotic organism.