ORC and Cdc6p interact and determine the frequency of initiation of DNA replication in the genome

Abstract

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.

Perpetuating the double helix: molecular machines at eukaryotic DNA replication origins

“…(27) Later, it was identified in Schizosaccharomyces pombe as a key regulator of chromosome ploidy (28) and again in S. cerevisiae as a suppressor of ORC mutations. (29) CDC6 is conserved in all eukarya and it encodes an AAA þ protein with significant sequence similarity to Orc1. (30) Its participation in initiation of DNA replication has been confirmed genetically and biochemically.…”

Roles of the CDK Phosphorylation Sites of Yeast Cdc6 in Chromatin Binding and Rereplication

“…The loading of Cdc6 onto chromatin is a critical step in the formation of a pre-RC (Liang et al, 1995;Cocker et al, 1996;Detweiler and Li, 1997). Thus, because Cdc6* reloads prematurely, strains expressing Cdc6* might be prone to rereplication.…”

“…In the yeast Saccharomyces cerevisiae, but also in many other organisms, the binding of Cdc6 to the origin recognition complex (ORC) is a critical step in the formation of pre-RCs (Liang et al, 1995;Cocker et al, 1996;Detweiler and Li, 1997) and essential for subsequent loading of Mcm proteins (Mcm2-7; Piatti et al, 1996;Santocanale and Diffley, 1996;Aparicio et al, 1997;Donovan et al, 1997;Tanaka et al, 1997). After licensing the origin by loading Mcms, endogenous Cdc6 dissociates from the replicative complex and only reassociates with chromatin late in M-phase Weinreich et al, 1999) when cyclin-dependent protein kinase (Clb-Cdc28) activities are absent.…”

Synthesis and turn‐over of the replicative Cdc6 protein during the HeLa cell cycle

“…In yeasts, Cdc6p is expressed during the G1 phase [7,8], associates with stationary ORC [9,10] and loads Mcm initiation proteins in reactions requiring an intact nucleotide binding domain [11][12][13]. Once replication begins, yeast Cdc6p is phosphorylated and then rapidly destroyed by ubiquitin-mediated protein degradation.…”

“…Cdc6p associates with the chromatin-bound six-subunit origin recognition complex (ORC) and promotes, together with the Cdt1 protein [2,3], the subsequent loading of the Mcm protein complex. The fully assembled pre-replicative complex is induced to activate replication origins by at least two classes of protein phosphorylating enzymes, cyclindependent kinases (Cdk) and the Dbf4-Cdc7 kinase [4][5][6].In yeasts, Cdc6p is expressed during the G1 phase [7,8], associates with stationary ORC [9,10] and loads Mcm initiation proteins in reactions requiring an intact nucleotide binding domain [11][12][13]. Once replication begins, yeast Cdc6p is phosphorylated and then rapidly destroyed by ubiquitin-mediated protein degradation.…”