The spatial arrangement of ORC binding modules determines the functionality of replication origins in budding yeast.
In the quest to define autonomously replicating sequences (ARSs) in eukaryotic cells, an ARS consensus sequence (ACS) has emerged for budding yeast. This ACS is recognized by the replication initiator, the origin recognition complex (ORC). However, not every match to the ACS constitutes a replication origin. Here, we investigated the requirements for ORC binding to origins that carry multiple, redundant ACSs, such as ARS603. Previous studies raised the possibility that these ACSs function as individual ORC binding sites. Detailed mutational analysis of the two ACSs in ARS603 revealed that they function in concert and give rise to an initiation pattern compatible with a single bipartite ORC binding site. Consistent with this notion, deletion of one base pair between the ACS matches abolished ORC binding at ARS603. Importantly, loss of ORC binding in vitro correlated with the loss of ARS activity in vivo. Our results argue that replication origins in yeast are in general comprised of bipartite ORC binding sites that cannot function in random alignment but must conform to a configuration that permits ORC binding. These requirements help to explain why only a limited number of ACS matches in the yeast genome qualify as ORC binding sites.
Bolon
Yung-Tsi
YT
Department of Biochemistry, Molecular Biology and Biophysics, 6-155 Jackson Hall, 321 Church Street SE, Minneapolis, MN 55455, USA.
Bielinsky
Anja-Katrin
AK
D001483:Base Sequence; D001665:Binding Sites; D016384:Consensus Sequence; D004261:DNA Replication; D004271:DNA, Fungal / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D051738:Origin Recognition Complex / Q000378:metabolism*; D011485:Protein Binding; D018741:Replication Origin; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*
D004271:DNA, Fungal; D051738:Origin Recognition Complex; D029701:Saccharomyces cerevisiae Proteins