ABSTRACT
We recently reported that the 'hydrophobic patch' (HP) of the Saccharomyces cerevisiae S-phase cyclin Clb5 facilitates its interaction with Orc6 (via its Cy or RXL motif), providing a mechanism that helps prevent rereplication from individual origins. This is the first finding of a biological function for an interaction between a cyclin and a cyclin-binding motif (Cy or RXL motif) in a target protein in Saccharomyces cerevisiae. It is also the first such example involving a B-type cyclin in any organism. Yet, some of our observations as well as work from other groups suggest that HP-RXL interactions are functionally important for cyclin-Cdk signaling to other targets. The evolutionary conservation of the HP motif suggests that it allows cyclins to carry out important and specialized functions.
Subject(s)
Cyclin B/physiology , Gene Expression Regulation, Fungal , Origin Recognition Complex/physiology , Saccharomyces cerevisiae Proteins/physiology , Saccharomyces cerevisiae/physiology , Amino Acid Motifs , Amino Acid Sequence , Cell Cycle/genetics , Cell Cycle/physiology , Cyclin B/chemistry , Cyclin B/genetics , Cyclin-Dependent Kinases/genetics , Cyclin-Dependent Kinases/physiology , Cyclins/genetics , Cyclins/physiology , DNA Replication , Hydrophobic and Hydrophilic Interactions , Molecular Sequence Data , Origin Recognition Complex/chemistry , Origin Recognition Complex/genetics , Protein Structure, Tertiary , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Signal Transduction/genetics , Signal Transduction/physiologyABSTRACT
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.
Subject(s)
Cyclin B/metabolism , DNA-Binding Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Amino Acid Sequence , Binding Sites , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cyclin B/chemistry , Cyclin B/genetics , DNA Replication , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Enzyme Stability , Mutation , Origin Recognition Complex , S Phase , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Two-Hybrid System TechniquesABSTRACT
Unrestrained E2F activity forces S phase entry and promotes apoptosis through p53-dependent and -independent mechanisms. Here, we show that deregulation of E2F by adenovirus E1A, loss of Rb or enforced E2F-1 expression results in the accumulation of caspase proenzymes through a direct transcriptional mechanism. Increased caspase levels seem to potentiate cell death in the presence of p53-generated signals that trigger caspase activation. Our results demonstrate that mitogenic oncogenes engage a tumour suppressor network that functions at multiple levels to efficiently induce cell death. The data also underscore how cell cycle progression can be coupled to the apoptotic machinery.
