ORC and Cdc6p interact and determine the frequency of initiation of DNA replication in the genome.
The origin recognition complex (ORC) binds replicators in the yeast S. cerevisiae in a manner consistent with it being an initiator protein for DNA replication. Two-dimensional (2D) gel techniques were used to examine directly initiation of chromosomal DNA replication in temperature-sensitive orc mutants. Unlike in wild-type cells, in orc2-1 and orc5-1 mutant cells, only a subset of replicators formed active origins of DNA replication at the permissive temperature. At the restrictive temperature, the number of active replicators was diminished further. Using a genetic screen, CDC6 was identified as a multicopy suppressor of orc5-1. 2D gel and biochemical analyses demonstrated that Cdc6p interacted functionally and physically with ORC. We suggest that ORC and Cdc6p form a prereplication complex at individual replicators and therefore cooperate to determine the frequency of initiation of DNA replication in the genome.
Liang
C
C
Cold Spring Harbor Laboratory, New York 11724, USA.
Weinreich
M
M
Stillman
B
B
D018797:Cell Cycle Proteins / Q000378:metabolism*; D015825:Chromosomes, Fungal; D003001:Cloning, Molecular; D004261:DNA Replication; D004271:DNA, Fungal / Q000096:biosynthesis; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D005804:Genes, Lethal; D016681:Genome, Fungal; D009154:Mutation; D051738:Origin Recognition Complex; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D018741:Replication Origin; D012097:Repressor Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D012641:Selection, Genetic; D013489:Suppression, Genetic
C066503:CDC6 protein, S cerevisiae; D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; C497215:ORC2 protein, S cerevisiae; C497220:ORC5 protein, S cerevisiae; D051738:Origin Recognition Complex; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins