A DNA UNWINDING ELEMENT AND AN ARS CONSENSUS COMPRISE A REPLICATION ORIGIN WITHIN A YEAST CHROMOSOME

We have defined a replication origin, ORI305, within chromosome III of Saccharomyces cerevisiae by means of mutational analysis. cis-acting elements required, for origin activity in the chromosome, as assayed b y two-dimensional gel electrophoresis of replication intermediates, ar e the same as those required for the function of an autonomously repli cating sequence, ARS305, in a plasmid. Essential elements include (i) an 11 bp sequence that is a near match to the ARS consensus and (ii) a broad sequence directly 3' to the consensus near match. Origin functi on is inactivated by point mutations in the essential near match seque nce, suggesting that the sequence contributes to specifying the origin in the chromosome. Other consensus near matches with different sequen ces are present but are not required. The essential 3'-flanking, seque nce exhibits DNA helical instability and is sensitive to deletion muta tions that stabilize the DNA helix. The wild-type 3'-flanking sequence can be functionally substituted by dissimilar sequences that also exh ibit helical instability. The requirement for DNA helical instability indicates that the essential 3'-flanking sequence serves as a DNA unwi nding element in the chromosome.