Cell Cycle Group

Our group studies chromosome replication and cytokinesis in eukaryotic cells. During 2005 we identified the Replisome Progression Complex (or RPC) that controls the advance of eukaryotic DNA replication forks. The RPC is built around the MCM helicase at origins during the initiation of chromosome replication. The four-protein GINS complex is a key component of the RPC as it allows other important factors to associate with MCM; these comprise Cdc45 that is thought to activate the MCM helicase, Tof1 and Csm3 that cause the replisome to pause upon encountering tight protein-DNA complexes, Mrc1 that allows checkpoint activation in response to problems in DNA synthesis, and Ctf4 that promotes the establishment of cohesion between the newly formed sister chromatids. The RPC also contains a histone chaperone that may facilitate the passage of the replisome through chromatin. In parallel with this work, we are also investigating how Cyclin-Dependent Kinase (CDK) represses cytokinesis until the end of mitosis.

The role of GINS during the initiation of chromosome replication
We carried out a systematic analysis of all the essential budding yeast proteins of previously unknown function in order to identify novel cell cycle factors. We generated yeast strains in which we modified each of the corresponding genes so that the encoded protein carried the 'heat-inducible degron' cassette at the amino terminus. This allowed us to induce rapid degradation of the target protein and then examine the immediate consequences. In this way we identified four proteins required for chromosome replication; remarkably these four proteins interact with each other to form a complex called GINS. During 2005, we showed that GINS is essential for the activation of 'pre-Replication Complexes' (or pre-RCs) that are built at every origin of DNA replication during the G1-phase of the cell cycle. The pre-RC contains an inactive form of the MCM helicase; activation occurs subsequently during S-phase when two kinases, Cdc7 and CDK, promote the recruitment of GINS to origins. GINS allows MCM to associate stably with another protein, Cdc45, which is thought to be an essential component of the active helicase. Loading of Cdc45 and GINS is a complicated process that also requires another factor Sld3. We showed that Sld3 is displaced from the origin during the loading reaction, indicating that it only acts during initiation, whereas GINS and Cdc45 then travel with MCM as part of the nascent replisome.

Building the Replisome Progression Complex
We purified GINS from yeast extracts and used mass spectrometry to show that it interacts with MCM and a group of other factors that mediate or control the progression of DNA replication forks. We found that all these proteins assemble around MCM during the establishment of DNA replication forks, to form what we call the 'Replisome Progression Complex'. GINS is essential for assembly of the RPC, as it allows a specific set of proteins to associate stably with MCM. These include Cdc45, and GINS is thus essential for unwinding of the origin and for activation of the pre-RC. In addition, GINS is needed for Mrc1, Tof1, Csm3 and Ctf4 to be incorporated into the RPC. The role of Mrc1, Tof1 and Csm3 is discussed below. Ctf4 is required for establishment of cohesion between the newly formed sister-chromatids, though the mechanism remains to be established.

Unwinding of the parental DNA duplex at forks causes the accumulation of positive supercoils that block further progression and must therefore be removed. We have shown that GINS allows the RPC to recruit Topoisomerase I to forks; it thus appears that Topoisomerase I normally carrys out this important role ahead of the replisome. Progression of the fork also requires 'unpacking' of chromatin so that the replisome can gain access to the chromosomal DNA. We have shown that the RPC contains a two-subunit histone chaperone called 'FACT' (Facilitates Chromatin Transcription) that is likely to be essential for progression of the replisome past nucleosomes, though this remains to be demonstrated directly.

The regulation of paused eukaryotic replisomes
DNA replication forks pause upon encountering tight non-nucleosomal protein-DNA complexes throughout the genome. We have generated a yeast strain in which efficient pausing can be induced at specific chromosomal loci, allowing us to study the nature and regulation of the paused replisome. We found that the RPC components Tof1 and Csm3 are essential for pausing of DNA replication forks, which is therefore a property of the paused replisome itself. In contrast, Mrc1 is not required for pausing, and is instead required for the activation of a checkpoint response when DNA synthesis is inhibited or defective. We have shown that a stable replisome remains associated with the paused fork, comprising the RPC together with DNA polymerases alpha and epsilon.

Cdc104 is essential for cytokinesis and is negatively regulated by CDK
We identified Cdc104 in our degron screen as a novel factor that is essential for cytokinesis. We are now studying the function and regulation of Cdc104 and have shown that it forms part of the actomyosin ring that assembles at the future site of cell division; contraction of the ring at the end of mitosis is an essential requirement for cytokinesis. Cdc104 is required for a very late step in cytokinesis, and only associates with the ring when CDK is inactivated at the end of anaphase.