John Diffley

Cyclin Dependent Kinases and DNA Replication in Yeast and Human Cells

We are interested in understanding the mechanism and regulation of eukaryotic DNA replication. We use budding yeast to study this process and have begun some parallel studies in mammalian cells.

DNA replication begins with the assembly of a pre-replicative complex (pre-RC) at future origins of replication. This involves the sequential binding of the origin recognition complex (ORC), Cdc6 and Cdt1-Mcm2-7. This process is inhibited in yeast by cyclin-dependent kinases (CDKs) and, as a consequence, can only occur during G1 phase when CDK activity is low.

CDKs inhibit pre-RC assembly by inhibiting each of the pre-RC components directly. Phosphorylation of Cdc6 targets it for ubiquitin-mediated proteolysis during S phase. Later in the cell cycle, phosphorylation of Cdc6 by CDKs generates a binding site for the mitotic cyclin Clb2 itself (see project 2 below).

The binding of Clb2 to Cdc6 inhibits its ability to interact with ORC. CDK activation at the end of G1 phase is then required to assemble additional factors, Cdc45 and GINS, onto the pre-RC. This assembly involves three essential factors, Sld2, Sld3 and Dpb11.

We have recently shown that Sld2 and Sld3 are the minimal set of CDK substrates required to trigger DNA replication. Their phosphorylation generates binding sites for tandem BRCT repeats in Dpb11 (see project 1 below). Using 'bypass' mutants in these proteins, we were able to induce DNA replication in the absence of CDK activity. There are two projects available:

1. Mammalian Sld3 Orthologues
   Sld3 is a poorly conserved, serine/threonine-rich protein. Identification of an Sld3 orthologue would allow us to begin to understand replication control by CDKs in mammalian cells. Although Sld3 is poorly conserved, the Dpb11 orthologue (TopBP1) has been identified.
   
   We will attempt to identify the Sld3 orthologue by looking for proteins that bind specifically and in a CDK-dependent manner to the N-terminal BRCT repeats from TopBP1. Once identified, we will employ analogous approaches to those used in yeast to determine if Sld3 is an essential CDK substrate and if Sld3 and Sld2 are the minimal set of CDK substrates required for replication in human cells.



2. Identification of Novel Clb2-binding Proteins
   Our previous work has shown that mitotic cyclins from both yeast and human cells bind specifically and in a phosphorylation-dependent manner to yeast Cdc6 protein. We believe that this phosphopeptide binding activity is a general strategy for substrate targeting by mitotic cyclins. To identify such substrates, we will use purified, recombinant CDKs to pull binding partners out of extracts (both yeast and human) and test the phosphorylation-dependence of this binding by comparing the binding before and after treatment with phosphatase.
   
   We will also purify endogenous Clb2 from yeast extracts under gentle conditions using tandem affinity purification. Associated proteins will be identified by mass spectrometry. In parallel we will generate Clb2 mutants that have lost the ability to bind phosphopeptdes to characterise the importance of this activity.

References

Mailand N and Diffley JFX. CDKs promote DNA replication origin licensing in human cells by protecting Cdc6 from APC/C¿dependent proteolysis. Cell 2005; 122: 915-926.

Mimura S, Seki T, et al. Phosphorylation-dependent binding of mitotic cyclins to Cdc6 contributes to DNA replication control. Nature 2004; 431: 1118-1123.

Zegerman P and Diffley JFX. Phosphorylation of Sld2 and Sld3 by cyclin-dependent kinases promotes DNA replication in budding yeast. Nature 2007; 445(7125): 281-285.