Karim Labib

GINS maintains association of Cdc45 with MCM in replisome progression complexes at eukaryotic DNA replication forks

The components of the replisome that preserve genomic stability by controlling the progression of eukaryotic DNA replication forks are poorly understood. Here, we show that the GINS (go ichi ni san) complex allows the MCM (minichromosome maintenance) helicase to interact with key regulatory proteins in large replisome progression complexes (RPCs) that are assembled during initiation and disassembled at the end of S phase. RPC components include the essential initiation and elongation factor, Cdc45, the checkpoint mediator Mrc1, the Tof1–Csm3 complex that allows replication forks to pause at protein–DNA barriers, the histone chaperone FACT (facilitates chromatin transcription) and Ctf4, which helps to establish sister chromatid cohesion. RPCs also interact with Mcm10 and topoisomerase I. During initiation, GINS is essential for a specific subset of RPC proteins to interact with MCM. GINS is also important for the normal progression of DNA replication forks, and we show that it is required after initiation to maintain the association between MCM and Cdc45 within RPCs.

Functional proteomic identification of DNA replication proteins by induced proteolysis in vivo

Evolutionarily diverse eukaryotic cells share many conserved proteins of unknown function. Some are essential for cell viability, emphasising their importance for fundamental processes of cell biology but complicating their analysis. We have developed an approach to the large-scale characterization of such proteins, based on conditional and rapid degradation of the target protein in vivo, so that the immediate consequences of bulk protein depletion can be examined. Budding yeast strains have been constructed in which essential proteins of unknown function have been fused to a ‘heat-inducible-degron’ cassette that targets the protein for proteolysis at 37 °C (ref. 4). By screening the collection for defects in cell-cycle progression, here we identify three DNA replication factors that interact with each other and that have uncharacterized homologues in human cells. We have used the degron strains to show that these proteins are required for the establishment and normal progression of DNA replication forks. The degron collection could also be used to identify other, essential, proteins with roles in many other processes of eukaryotic cell biology.

How do Cdc7 and cyclin-dependent kinases trigger the initiation of chromosome replication in eukaryotic cells?

Chromosome replication occurs precisely once during the cell cycle of almost all eukaryotic cells, and is a highly complex process that is still understood relatively poorly. Two conserved kinases called Cdc7 (cell division cycle 7) and cyclin-dependent kinase (CDK) are required to establish replication forks during the initiation of chromosome replication, and a key feature of this process is the activation of the replicative DNA helicase in situ at each origin of DNA replication. A series of recent studies has shed new light on the targets of Cdc7 and CDK, indicating that chromosome replication probably initiates by a fundamentally similar mechanism in all eukaryotes.

A key role for Ctf4 in coupling the MCM2-7 helicase to DNA polymerase α within the eukaryotic replisome

The eukaryotic replisome is a crucial determinant of genome stability, but its structure is still poorly understood. We found previously that many regulatory proteins assemble around the MCM2‐7 helicase at yeast replication forks to form the replisome progression complex (RPC), which might link MCM2‐7 to other replisome components. Here, we show that the RPC associates with DNA polymerase α that primes each Okazaki fragment during lagging strand synthesis. Our data indicate that a complex of the GINS and Ctf4 components of the RPC is crucial to couple MCM2‐7 to DNA polymerase α. Others have found recently that the Mrc1 subunit of RPCs binds DNA polymerase epsilon, which synthesises the leading strand at DNA replication forks. We show that cells lacking both Ctf4 and Mrc1 experience chronic activation of the DNA damage checkpoint during chromosome replication and do not complete the cell cycle. These findings indicate that coupling MCM2‐7 to replicative polymerases is an important feature of the regulation of chromosome replication in eukaryotes, and highlight a key role for Ctf4 in this process.

Distinct roles for Sld3 and GINS during establishment and progression of eukaryotic DNA replication forks

The Cdc45 protein is crucial for the initiation of chromosome replication in eukaryotic cells, as it allows the activation of prereplication complexes (pre‐RCs) that contain the MCM helicase. This causes the unwinding of origins and the establishment of DNA replication forks. The incorporation of Cdc45 at nascent forks is a highly regulated and poorly understood process that requires, in budding yeast, the Sld3 protein and the GINS complex. Previous studies suggested that Sld3 is also important for the progression of DNA replication forks after the initiation step, as are Cdc45 and GINS. In contrast, we show here that Sld3 does not move with DNA replication forks and only associates with MCM in an unstable manner before initiation. After the establishment of DNA replication forks from early origins, Sld3 is no longer essential for the completion of chromosome replication. Unlike Sld3, GINS is not required for the initial recruitment of Cdc45 to origins and instead is necessary for stable engagement of Cdc45 with the nascent replisome. Like Cdc45, GINS then associates stably with MCM during S‐phase.

Replication fork barriers: pausing for a break or stalling for time?

Defects in chromosome replication can lead to translocations that are thought to result from recombination events at stalled DNA replication forks. The progression of forks is controlled by an essential DNA helicase, which unwinds the parental duplex and can stall on encountering tight protein–DNA complexes. Such pause sites are hotspots for recombination and it has been proposed that stalled replisomes disassemble, leading to fork collapse. However, in both prokaryotes and eukaryotes it now seems that paused forks are surprisingly stable, so that DNA synthesis can resume without recombination if the barrier protein is removed. Recombination at stalled forks might require other events that occur after pausing, or might be dependent on features of the surrounding DNA sequence. These findings have important implications for our understanding of the regulation of genome stability in eukaryotic cells, in which pausing of forks is mediated by specific proteins that are associated with the replicative helicase.

Replisome stability at defective DNA replication forks is independent of S phase checkpoint kinases.

The S phase checkpoint pathway preserves genome stability by protecting defective DNA replication forks, but the underlying mechanisms are still understood poorly. Previous work with budding yeast suggested that the checkpoint kinases Mec1 and Rad53 might prevent collapse of the replisome when nucleotide concentrations are limiting, thereby allowing the subsequent resumption of DNA synthesis. Here we describe a direct analysis of replisome stability in budding yeast cells lacking checkpoint kinases, together with a high-resolution view of replisome progression across the genome. Surprisingly, we find that the replisome is stably associated with DNA replication forks following replication stress in the absence of Mec1 or Rad53. A component of the replicative DNA helicase is phosphorylated within the replisome in a Mec1-dependent manner upon replication stress, and our data indicate that checkpoint kinases control replisome function rather than stability, as part of a multifaceted response that allows cells to survive defects in chromosome replication.

Mcm10 associates with the loaded DNA helicase at replication origins and defines a novel step in its activation

Mcm10 is essential for chromosome replication in eukaryotic cells and was previously thought to link the Mcm2‐7 DNA helicase at replication forks to DNA polymerase alpha. Here, we show that yeast Mcm10 interacts preferentially with the fraction of the Mcm2‐7 helicase that is loaded in an inactive form at origins of DNA replication, suggesting a role for Mcm10 during the initiation of chromosome replication, but Mcm10 is not a stable component of the replisome subsequently. Studies with budding yeast and human cells indicated that Mcm10 chaperones the catalytic subunit of polymerase alpha and preserves its stability. We used a novel degron allele to inactivate Mcm10 efficiently and this blocked the initiation of chromosome replication without causing degradation of DNA polymerase alpha. Strikingly, the other essential helicase subunits Cdc45 and GINS were still recruited to Mcm2‐7 when cells entered S‐phase without Mcm10, but origin unwinding was blocked. These findings indicate that Mcm10 is required for a novel step during activation of the Cdc45–MCM–GINS helicase at DNA replication origins.

A Ctf4 trimer couples the CMG helicase to DNA polymerase α in the eukaryotic replisome

Efficient duplication of the genome requires the concerted action of helicase and DNA polymerases at replication forks to avoid stalling of the replication machinery and consequent genomic instability. In eukaryotes, the physical coupling between helicase and DNA polymerases remains poorly understood. Here we define the molecular mechanism by which the yeast Ctf4 protein links the Cdc45–MCM–GINS (CMG) DNA helicase to DNA polymerase α (Pol α) within the replisome. We use X-ray crystallography and electron microscopy to show that Ctf4 self-associates in a constitutive disk-shaped trimer. Trimerization depends on a β-propeller domain in the carboxy-terminal half of the protein, which is fused to a helical extension that protrudes from one face of the trimeric disk. Critically, Pol α and the CMG helicase share a common mechanism of interaction with Ctf4. We show that the amino-terminal tails of the catalytic subunit of Pol α and the Sld5 subunit of GINS contain a conserved Ctf4-binding motif that docks onto the exposed helical extension of a Ctf4 protomer within the trimer. Accordingly, we demonstrate that one Ctf4 trimer can support binding of up to three partner proteins, including the simultaneous association with both Pol α and GINS. Our findings indicate that Ctf4 can couple two molecules of Pol α to one CMG helicase within the replisome, providing a new model for lagging-strand synthesis in eukaryotes that resembles the emerging model for the simpler replisome of Escherichia coli. The ability of Ctf4 to act as a platform for multivalent interactions illustrates a mechanism for the concurrent recruitment of factors that act together at the fork.

Cdc48 and a ubiquitin ligase drive disassembly of the CMG helicase at the end of DNA replication

Introduction Chromosome replication is initiated by a universal mechanism in eukaryotic cells. This mechanism entails the assembly and activation at replication origins of the DNA helicase known as CMG (Cdc45-MCM-GINS), which is essential for the progression of replication forks. The replisome is built around the CMG helicase, which associates stably with DNA replication forks until the termination of DNA synthesis. The mechanism by which CMG is disassembled was unknown until now but is likely to represent a key regulated step at the end of chromosome replication. Regulated disassembly of the CMG helicase at the end of chromosome replication in budding yeast. It is very important that the CMG helicase is not displaced from replication forks during elongation, because it cannot be reloaded. When replication terminates, however, the ubiquitin ligase SCFDia2 and the Cdc48 segregase induce disassembly of the CMG helicase, leading to dissolution of the replisome. Rationale The CMG helicase exists only at DNA replication forks but can be isolated from extracts of S-phase budding yeast cells, after digestion of chromosomal DNA. We screened for posttranslational modifications of the CMG helicase that might regulate its function. Results Here we show that the CMG helicase is ubiquitylated during the final stages of chromosome replication in Saccharomyces cerevisiae, specifically on its Mcm7 subunit. The F-box protein Dia2 is essential in vivo for ubiquitylation of CMG, and the SCFDia2 ubiquitin ligase is also required to ubiquitylate CMG in vitro on its Mcm7 subunit in extracts of S-phase yeast cells. Ubiquitylated CMG exists only transiently in vivo, as it is rapidly disassembled in a reaction that is independent of the proteasome but requires the Cdc48/p97 segregase, which associates with ubiquitylated CMG. Consistent with these data, we show that Dia2 is essential for disassembly of the CMG helicase at the end of S phase in budding yeast. Rather than causing dissolution of the active helicase, Dia2 specifically induces the disassembly of terminated CMG complexes, which suggests that the helicase undergoes a change at the end of DNA replication, predisposing it for disassembly. Conclusion Our findings indicate that the end of chromosome replication in eukaryotes is controlled in a similarly complex fashion to the much-better-characterized initiation step. Our findings indicate that replisome disassembly is driven by the regulated dissolution of the stable CMG helicase, which uses its hexameric ring of Mcm2-7 proteins to encircle the parental DNA at replication forks. Our data identify two key features of helicase disassembly in budding yeast: First, there is an essential role for the F-box protein Dia2, which drives ubiquitylation of the CMG helicase on its Mcm7 subunit. Second, the Cdc48 segregase is required to break ubiquitylated CMG into its component parts. Once separated from GINS and Cdc45, the Mcm2-7 hexamer is less stable, so that all of the subunits of the CMG helicase are lost from the newly replicated DNA. Just as the main features of helicase assembly have been conserved across evolution from yeasts to humans, we envisage that disassembly of the CMG helicase will also involve a universal mechanism in eukaryotic cells, by which Cdc48/p97 drives disassembly of ubiquitylated CMG at the end of DNA replication. How to stop after copying the genome Replication is highly regulated: Failure to copy any part of the genome or copying parts of it more than once can cause genome instability with potentially disastrous consequences. Maric et al. and Priego Moreno et al. show that the DNA replication machinery, which stably encircles DNA during the duplication process, is actively disassembled once replication is complete (see the Perspective by Bell). The protein ring encircling the DNA is covalently modified, which allows it to be opened and the whole replication complex to be removed from DNA by a special disassembly complex. Science, this issue 10.1126/science.1253596, p. 477; see also p. 418 DNA replication machinery stably encircles replicating DNA and is actively disassembled once replication is complete. [Also see Perspective by Bell] Chromosome replication is initiated by a universal mechanism in eukaryotic cells, involving the assembly and activation at replication origins of the CMG (Cdc45-MCM-GINS) DNA helicase, which is essential for the progression of replication forks. Disassembly of CMG is likely to be a key regulated step at the end of chromosome replication, but the mechanism was unknown until now. Here we show that the ubiquitin ligase known as SCFDia2 promotes ubiquitylation of CMG during the final stages of chromosome replication in Saccharomyces cerevisiae. The Cdc48/p97 segregase then associates with ubiquitylated CMG, leading rapidly to helicase disassembly. These findings indicate that the end of chromosome replication in eukaryotes is controlled in a similarly complex fashion to the much-better-characterized initiation step.