INITIATION PREFERENCE AT A YEAST ORIGIN OF REPLICATION

Replication origins in the yeast Saccharomyces cerevisiae are identifi ed as autonomous replication sequence (ARS) elements. To examine the e ffect of origin density on replication initiation, we have analyzed th e replication of a plasmid that contains two copies of the same origin , ARS1. The activation of origins and the direction that replication f orks move through flanking sequences can be physically determined by a nalyzing replication intermediates on two-dimensional agarose gels. We find that only one of the two identical ARSs on the plasmid initiates replication on any given plasmid molecule; that is, this close spacin g of ARSs results in an apparent interference between the potential or igins. Moreover, in the particular plasmid that we constructed, one of the two identical copies of ARS1 is used four times more frequently t han the other one. These results show that the plasmid context is crit ical for determining the preferred origin. This origin preference is a lso exhibited when the tandem copies of ARS1 are introduced into a yea st chromosome. The sequences responsible for establishing the origin p reference have been identified by deletion analysis and are found to r eside in a portion of the yeast URA3 gene.