INITIATION COMPLEX ASSEMBLY AT BUDDING YEAST REPLICATION ORIGINS BEGINS WITH THE RECOGNITION OF A BIPARTITE SEQUENCE BY LIMITING AMOUNTS OFTHE INITIATOR, ORC

Characterization of the proteins that interact with replication origin s, as well as characterization of the mechanisms by which the levels a nd activities of these proteins are regulated during the cell cycle, i s required to understand the initiation of chromosomal DNA replication in eukaryotic cells. We have previously shown that the first detectab le step in the assembly of initiation complexes in vivo involves the b inding of the multisubunit origin recognition complex (ORC) and the ge neral transcription/replication factor ABF1 protein to origins. In thi s paper we show that ORC is present in cells at low levels, correspond ing to little more than one complete complex per replication origin, i ndicating that in vivo origin recognition by ORC is extremely efficien t, We show that this efficient recognition requires two sequence eleme nts, the essential A element containing the ARS consensus sequence and the functionally important B1 element, both in vitro and in vivo. Mor eover, we show that origin binding by ORC in vivo does not require any other functional sequence element, indicating that it occurs independ ently of the binding of other factors, such as ABF1, Our results sugge st a model for the roles of the individual elements of yeast replicati on origins.