Initiation complex assembly at budding yeast replication origins begins with the recognition of a bipartite sequence by limiting amounts of the initiator, ORC.
Characterization of the proteins that interact with replication origins, as well as characterization of the mechanisms by which the levels and activities of these proteins are regulated during the cell cycle, is required to understand the initiation of chromosomal DNA replication in eukaryotic cells. We have previously shown that the first detectable step in the assembly of initiation complexes in vivo involves the binding of the multisubunit origin recognition complex (ORC) and the general transcription/replication factor ABF1 protein to origins. In this paper we show that ORC is present in cells at low levels, corresponding to little more than one complete complex per replication origin, indicating that in vivo origin recognition by ORC is extremely efficient. We show that this efficient recognition requires two sequence elements, the essential A element containing the ARS consensus sequence and the functionally important B1 element, both in vitro and in vivo. Moreover, we show that origin binding by ORC in vivo does not require any other functional sequence element, indicating that it occurs independently of the binding of other factors, such as ABF1. Our results suggest a model for the roles of the individual elements of yeast replication origins.
Rowley
A
A
Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hertfordshire, UK.
Cocker
J H
JH
Harwood
J
J
Diffley
J F
JF
D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D017931:DNA Primers / Q000235:genetics; D004261:DNA Replication; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism; D008957:Models, Genetic; D008969:Molecular Sequence Data; D051738:Origin Recognition Complex; D011485:Protein Binding; D018741:Replication Origin; D012097:Repressor Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*
D017931:DNA Primers; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D051738:Origin Recognition Complex; D012097:Repressor Proteins