MULTIPLE DNA ELEMENTS IN ARS305 DETERMINE REPLICATION ORIGIN ACTIVITYIN A YEAST CHROMOSOME

A yeast autonomously replicating sequence, ARS305, shares essential co mponents with a chromosome III replicator, ORI305, Known components in clude an ARS consensus sequence (ACS) element, presumed to bind the or igin recognition complex (ORC), and a broad 3'-flanking sequence which contains a DNA unwinding element, Here linker substitution mutagenesi s of ARS305 and analysis of plasmid mitotic stability identified three short sequence elements within the broad 3'-flanking sequence, The ma jor functional element resides directly 3' of the ACS and the two rema ining elements reside further downstream, all within non-conserved ARS sequences, To determine the contribution of the elements to replicati on origin function in the chromosome, selected linker mutations were t ransplaced into the ORI305 locus and two-dimensional gel electrophores is was used to analyze replication bubble formation and fork direction s. Mutation of the major functional element identified in the plasmid mitotic stability assay inactivated replication origin function in the chromosome, Mutation of each of the two remaining elements diminished both plasmid ARS and chromosomal origin activities to similar levels, Thus multiple DIVA elements identified in the plasmid ARS are determi nants of replication origin function in the natural context of the chr omosome, Comparison with two other genetically defined chromosomal rep licators reveals a conservation of functional elements known to bind O RC, but no two replicators are identical in the arrangement of element s downstream of ORC binding elements or in the extent of functional se quences adjacent to the ACS.