Interaction of the S-phase cyclin Clb5 with an 'RXL' docking sequence in the initiator protein Orc6 provides an origin-localized replication control switch

Abstract

Cyclin-dependent kinases are critical regulators of eukaryotic DNA replication. We show that the S-phase cyclin Clb5 binds stably and directly to the origin recognition complex (ORC). This interaction is mediated by an "RXL" target sequence, or "Cy" motif, in the Orc6 subunit that is recognized by the "hydrophobic patch" region on Clb5. The Clb5-Orc6 interaction requires replication initiation, and is maintained throughout the remainder of S phase and into M phase. Eliminating the Clb5-Orc6 interaction has no effect on initiation of replication but instead sensitizes cells to lethal overreplication. We propose that Clb5 binding to ORC provides an origin-localized replication control switch that specifically prevents reinitiation at replicated origins.[Keywords: Cell cycle control; DNA replication; cyclin-dependent kinase; origin recognition complex; genomic stability; re-replication] Supplemental material is available at http://www.genesdev.org. The eukaryotic cell cycle is controlled by oscillations in cyclin-dependent kinase (Cdk) activity (Zachariae and Nasmyth 1999). Cdk activity oscillations are required because critical cell cycle steps are both positively and negatively regulated by Cdk activity. A well-characterized example is DNA replication (Bell and Dutta 2002), in which oscillating Cdk levels control the formation and activation of protein complexes at origins of DNA replication.In budding yeast, origins are bound throughout the cell cycle by the six-member origin recognition complex (ORC;Diffley et al. 1994). As cells pass from the M to the G1 phase of the cell cycle, Cdc6 and Cdt1 proteins interact with the ORC-bound origin and direct the loading of the six-member Mcm2-7 complex (Cocker et al. 1996;Aparicio et al. 1997;Tanaka et al. 1997;Devault et al. 2002;Tanaka and Diffley 2002). The resulting structure is called the "pre-Replicative Complex" or pre-RC.As cells enter S phase, elevated levels of Cdk activity stimulate initiation of DNA replication from the pre-RC loaded origins (Schwob et al. 1994). Positive control of replication by Cdk activity in Saccharomyces cerevisiae is primarily mediated by S-phase cyclins Clb5 and Clb6 (Schwob and Nasmyth 1993;Schwob et al. 1994;Donaldson et al. 1998a;Donaldson 2000; Epstein and Cross 2002), although it is not well understood how Cdk activity induces replication. Cdk phosphorylation of the Sld2/Drc1 replication protein has been demonstrated to be essential for replication initiation (Masumoto et al. 2002), but there may be additional activating substrates. During S phase, ORC is normally phosphorylated by Clb5,6, and biochemical studies have identified an interaction between Clb5 and ORC (Weinreich et al. 2001). The function of this interaction has been unclear. In Xenopus, cyclin E is recruited to origins of replication through Cdc6 to activate replication (Furstenthal et al. 2001a,b), although the targets for cyclin E-directed Cdk activity are also unclear.

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

“…After 4 h, cells were fixed and stained with propidium iodide, and DNA contents were measured by flow cytometry. (Nguyen et al, 2001;Vas et al, 2001;Mimura et al, 2004;Wilmes et al, 2004), and interactions have been seen between alleles of ORC6 and CDC6 (Wilmes et al, 2004), but results differ in detail. Nguyen et al and Wilmes et al found that an ectopic NLS on Mcm7 was also required for rereplication, whereas we and Mimura et al saw no effect of Mcm7-NLS.…”

“…However, the experiments differed in at least two ways. First, Nguyen et al and Wilmes et al used Second, Nguyen et al (2001) and Wilmes et al (2004) found dependence of rereplication on Mcm7-NLS using flow cytometry to monitor DNA content (a relatively insensitive assay), whereas we and Mimura et al (2004) saw the lack of dependence on Mcm7-NLS in assays that measured cell viability (a probably more sensitive assay). Thus it may be that rereplication does not absolutely require Mcm7-NLS, but is more extensive (and therefore easier to see by flow cytometry) in its presence.…”

“…As an admittedly speculative model to explain how CDC6* could be redundant with ORC2* ORC6*, we propose the following: Rereplication is normally prevented because the cell localizes Clb5-Cdc28 to the origin via Orc6 (Wilmes et al, 2004), and redundantly (we propose), the cell localizes Clb2-Cdc28 (and Clb1-Cdc28) to the origin via Cdc6. Mimura et al (2004) showed binding between Cdc6 and Clb2, and we suggest that this binding might allow Cdc6 to bring Clb2-Cdc28 to the origin.…”

“…Once at the origin, both Clb5-Cdc28 and Clb2-Cdc28 phosphorylate a partially overlapping set of initiator proteins, preventing rereplication. Phosphorylation site mutants of Cdc6 (which fail to bind Clb2) are synthetically lethal with clb5 deletion mutants (Wilmes et al, 2004) because there is then no way to localize any Clb-CDK activity to the origin. Similarly, CDC6* is synthetically lethal with ORC2* ORC6* because Cdc6* cannot target Clb2 to the origin, and Orc2 and Orc6 are the most important targets of origin-localized Clb5-Cdc28 (and furthermore may aid in the binding of Clb5 to Orc).…”

“…Indeed, it has previously been shown (Nguyen et al, 2001;Vas et al, 2001;Mimura et al, 2004;Wilmes et al, 2004) that CDC6 mutants similar to CDC6* sensitize strains to rereplication. We looked for rereplication using our own CDC6 constructs, which are somewhat different from used previously.…”