• The amino-terminal TPR domain of Dia2 tethers SCF(Dia2) to the replisome progression complex.

      Morohashi, Hiroko; Maculins, Timurs; Labib, Karim; Cancer Research UK Paterson Institute for Cancer Research, University of Manchester, UK. (2009-12-01)
      Eukaryotic cells contain multiple versions of the E3 ubiquitin ligase known as the SCF (Skp1/cullin/F box), each of which is distinguished by a different F box protein that uses a domain at the carboxyl terminus to recognize substrates [1, 2]. The F box protein Dia2 is an important determinant of genome stability in budding yeast [3-5], but its mode of action is poorly understood. Here we show that SCF(Dia2) associates with the replisome progression complex (RPC) that assembles around the MCM2-7 helicase at DNA replication forks [6]. This interaction requires the RPC components Mrc1 and Ctf4, both of which associate with a tetratricopeptide repeat (TPR) domain located at the amino terminus of Dia2. Our data indicate that the TPR domain of Dia2 tethers SCF(Dia2) to the RPC, probably increasing the local concentration of the ligase at DNA replication forks. This regulation becomes important in cells that accumulate stalled DNA replication forks at protein-DNA barriers, perhaps aiding the interaction of SCF(Dia2) with key substrates. Our findings suggest that the amino-terminal domains of other F box proteins might also play an analogous regulatory role, controlling the localization of the cognate SCF complexes.
    • Building a double hexamer of DNA helicase at eukaryotic replication origins.

      Labib, Karim; Cell Cycle group, Paterson Institute for Cancer Research, University of Manchester, Manchester, UK. klabib@picr.man.ac.uk (2011-12-14)
    • The chromosome replication cycle.

      Diffley, John F X; Labib, Karim; Cancer Research UK, Lincoln's Inn Fields and Clare Hall Institute, Blanche Lane, South Mimms, EN6 3LD, UK. j.diffley@cancer.org.uk (2002-03-01)
    • A conserved motif in the C-terminal tail of DNA polymerase α tethers primase to the eukaryotic replisome.

      Kilkenny, M; De Piccoli, Giacomo; Perera, R; Labib, Karim; Pellegrini, L; Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, United Kingdom. (2012-07-06)
      The DNA polymerase α-primase complex forms an essential part of the eukaryotic replisome. The catalytic subunits of primase and pol α synthesize composite RNA-DNA primers that initiate the leading and lagging DNA strands at replication forks. The physical basis and physiological significance of tethering primase to the eukaryotic replisome via pol α remain poorly characterized. We have identified a short conserved motif at the extreme C terminus of pol α that is critical for interaction of the yeast ortholog pol1 with primase. We show that truncation of the C-terminal residues 1452-1468 of Pol1 abrogates the interaction with the primase, as does mutation to alanine of the invariant amino acid Phe(1463). Conversely, a pol1 peptide spanning the last 16 residues binds primase with high affinity, and the equivalent peptide from human Pol α binds primase in an analogous fashion. These in vitro data are mirrored by experiments in yeast cells, as primase does not interact in cell extracts with pol1 that either terminates at residue 1452 or has the F1463A mutation. The ability to disrupt the association between primase and pol α allowed us to assess the physiological significance of primase being tethered to the eukaryotic replisome in this way. We find that the F1463A mutation in Pol1 renders yeast cells dependent on the S phase checkpoint, whereas truncation of Pol1 at amino acid 1452 blocks yeast cell proliferation. These findings indicate that tethering of primase to the replisome by pol α is critical for the normal action of DNA replication forks in eukaryotic cells.
    • Distinct roles for Sld3 and GINS during establishment and progression of eukaryotic DNA replication forks.

      Kanemaki, Masato; Labib, Karim; Cancer Research UK. (2006-04-19)
      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.
    • Dpb2 integrates the leading-strand DNA polymerase into the eukaryotic replisome.

      Sengupta, Sugopa; Van Deursen, Frederick; de Piccoli, Giacomo; Labib, Karim; Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester M20 4BX, UK. (2013-04-08)
      The eukaryotic replisome is a critical determinant of genome integrity with a complex structure that remains poorly characterized. A central unresolved issue is how the Cdc45-MCM-GINS helicase is linked to DNA polymerase epsilon, which synthesizes the leading strand at replication forks and is an important focus of regulation.
    • Eukaryotic replisome components cooperate to process histones during chromosome replication.

      Foltman, Magdalena; Evrin, Cecile; De Piccoli, Giacomo; Jones, R; Edmondson, R; Katou, Y; Nakato, R; Shirahige, K; Labib, Karim; Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester M20 4BX, UK. (2013-03-28)
      DNA unwinding at eukaryotic replication forks displaces parental histones, which must be redeposited onto nascent DNA in order to preserve chromatin structure. By screening systematically for replisome components that pick up histones released from chromatin into a yeast cell extract, we found that the Mcm2 helicase subunit binds histones cooperatively with the FACT (facilitiates chromatin transcription) complex, which helps to re-establish chromatin during transcription. FACT does not associate with the Mcm2-7 helicase at replication origins during G1 phase but is subsequently incorporated into the replisome progression complex independently of histone binding and uniquely among histone chaperones. The amino terminal tail of Mcm2 binds histones via a conserved motif that is dispensable for DNA synthesis per se but helps preserve subtelomeric chromatin, retain the 2 micron minichromosome, and support growth in the absence of Ctf18-RFC. Our data indicate that the eukaryotic replication and transcription machineries use analogous assemblies of multiple chaperones to preserve chromatin integrity.
    • Functional proteomic identification of DNA replication proteins by induced proteolysis in vivo.

      Kanemaki, Masato; Sanchez-Diaz, Alberto; Gambus, Agnieszka; Labib, Karim; Cancer Research UK, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Manchester, M20 4BX, UK. (2003-06-12)
      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 degrees 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.
    • GINS maintains association of Cdc45 with MCM in replisome progression complexes at eukaryotic DNA replication forks.

      Gambus, Agnieszka; Jones, Richard C; Sanchez-Diaz, Alberto; Kanemaki, Masato; Van Deursen, Frederick; Edmondson, Ricky D; Labib, Karim; Cancer Research UK, Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester, M20 4BX, UK. (2006-04)
      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.
    • Hof1 and Rvs167 have redundant roles in actomyosin ring function during cytokinesis in budding yeast.

      Nkosi, Pedro Junior; Targosz, Bianca-Sabrina; Labib, Karim; Sanchez-Diaz, Alberto; Paterson Institute for Cancer Research, University of Manchester, Manchester, United Kingdom. (2013)
      The Hof1 protein (Homologue of Fifteen) regulates formation of the primary septum during cytokinesis in the budding yeast , whereas the orthologous Cdc15 protein in fission yeast regulates the actomyosin ring by using its F-BAR domain to recruit actin nucleators to the cleavage site. Here we show that budding yeast Hof1 also contributes to actin ring assembly in parallel with the Rvs167 protein. Simultaneous deletion of the and genes is lethal, and cells fail to assemble the actomyosin ring as they progress through mitosis. Although Hof1 and Rvs167 are not orthologues, they both share an analogous structure, with an F-BAR or BAR domain at the amino terminus, capable of inducing membrane curvature, and SH3 domains at the carboxyl terminus that bind to specific proline-rich targets. The SH3 domain of Rvs167 becomes essential for assembly of the actomyosin ring in cells lacking Hof1, suggesting that it helps to recruit a regulator of the actin cytoskeleton. This new function of Rvs167 appears to be independent of its known role as a regulator of the Arp2/3 actin nucleator, as actin ring assembly is not abolished by the simultaneous inactivation of Hof1 and Arp2/3. Instead we find that recruitment to the bud-neck of the Iqg1 actin regulator is defective in cells lacking Hof1 and Rvs167, though future studies will be needed to determine if this reflects a direct interaction between these factors. The redundant role of Hof1 in actin ring assembly suggests that the mechanism of actin ring assembly has been conserved to a greater extent across evolution than anticipated previously.
    • How do Cdc7 and cyclin-dependent kinases trigger the initiation of chromosome replication in eukaryotic cells?

      Labib, Karim; Cancer Research UK, Paterson Institute for Cancer Research, University of Manchester, Manchester M20 4BX, United Kingdom. klabib@picr.man.ac.uk (2010-06-15)
      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.
    • Inn1 couples contraction of the actomyosin ring to membrane ingression during cytokinesis in budding yeast.

      Sanchez-Diaz, Alberto; Marchesi, Vanessa; Murray, Stephen M; Jones, Richard C; Pereira, Gislene; Edmondson, Ricky D; Allen, Terence D; Labib, Karim; Cancer Research U.K., Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester, M20 4BX, UK. (2008-04)
      By rapidly depleting each of the essential budding yeast proteins of unknown function, we identified a novel factor that we call Inn1, which associates with the contractile actomyosin ring at the end of mitosis and is needed for cytokinesis. We show that Inn1 has a C2 domain at the amino terminus of the protein that is required for ingression of the plasma membrane, whereas the remainder of the protein recruits Inn1 to the actomyosin ring. The lethal effects of deleting the INN1 gene can be suppressed by artificial fusion of the C2 domain to other components of the actomyosin ring, restoring membrane ingression on contraction of the actomyosin ring. Our data indicate that recruitment of the C2 domain of Inn1 to the contractile actomyosin ring is crucial for ingression of the plasma membrane during cytokinesis in budding yeast.
    • A key role for Ctf4 in coupling the MCM2-7 helicase to DNA polymerase alpha within the eukaryotic replisome.

      Gambus, Agnieszka; Van Deursen, Frederick; Polychronopoulos, Dimitrios; Foltman, Magdalena; Jones, Richard C; Edmondson, Ricky D; Calzada, Arturo; Labib, Karim; Cancer Research UK, Paterson Institute for Cancer Research, University of Manchester, Manchester, UK. (2009-10-07)
      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 alpha 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 alpha. 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.
    • A key role for the GINS complex at DNA replication forks

      Labib, Karim; Gambus, Agnieszka; Cancer Research U.K., Paterson Institute for Cancer Research, University of Manchester, Manchester, UK. klabib@picr.man.ac.uk <klabib@picr.man.ac.uk> (2007-06)
      The GINS complex is the most recently identified component of the eukaryotic DNA replication machinery and is required both for the initiation of chromosome replication and also for the normal progression of DNA replication forks. Several recent studies suggest that GINS associates at replication forks with the MCM helicase that is responsible for unwinding the parental DNA duplex. Archaea also have an equivalent GINS complex that can interact with MCM and other replisome components. It seems likely that GINS couples MCM to other key proteins at forks, and we discuss here the current literature regarding this important late-comer to the DNA replication field.
    • Making connections at DNA replication forks: Mrc1 takes the lead.

      Labib, Karim; Cancer Research UK, Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester M20 4BX, UK. (2008-10-24)
      In a recent issue of Molecular Cell, Lou et al. (2008) demonstrate that the Mrc1 protein associates with the DNA polymerase that acts on the leading strand at replication forks, suggesting a potential mechanism that could help to preserve genome stability.
    • Mcm10 associates with the loaded DNA helicase at replication origins and defines a novel step in its activation.

      Van Deursen, Frederick; Sengupta, Sugopa; De Piccoli, Giacomo; Sanchez-Diaz, Alberto; Labib, Karim; Paterson Institute for Cancer Research, University of Manchester, Manchester, UK. (2012)
      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.
    • The Mitotic Exit Network and Cdc14 phosphatase initiate cytokinesis by counteracting CDK phosphorylations and blocking polarised growth.

      Sanchez-Diaz, Alberto; Nkosi, Pedro Junior; Murray, Stephen M; Labib, Karim; Paterson Institute for Cancer Research, University of Manchester, Manchester, UK. (2012-08-29)
      Polarisation of the actin cytoskeleton must cease during cytokinesis, to support efficient assembly and contraction of the actomyosin ring at the site of cell division, but the underlying mechanisms are still understood poorly in most species. In budding yeast, the Mitotic Exit Network (MEN) releases Cdc14 phosphatase from the nucleolus during anaphase, leading to the inactivation of mitotic forms of cyclin-dependent kinase (CDK) and the onset of septation, before G1-CDK can be reactivated and drive re-polarisation of the actin cytoskeleton to a new bud. Here, we show that premature inactivation of mitotic CDK, before release of Cdc14, allows G1-CDK to divert the actin cytoskeleton away from the actomyosin ring to a new site of polarised growth, thereby delaying progression through cytokinesis. Our data indicate that cells normally avoid this problem via the MEN-dependent release of Cdc14, which counteracts all classes of CDK-mediated phosphorylations during cytokinesis and blocks polarised growth. The dephosphorylation of CDK targets is therefore central to the mechanism by which the MEN and Cdc14 initiate cytokinesis and block polarised growth during late mitosis.
    • Molecular anatomy and regulation of a stable replisome at a paused eukaryotic DNA replication fork.

      Calzada, Arturo; Hodgson, Ben; Kanemaki, Masato; Bueno, Avelino; Labib, Karim; Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, UK. (2005-08-15)
      Eukaryotic cells regulate the progression and integrity of DNA replication forks to maintain genomic stability and couple DNA synthesis to other processes. The budding yeast proteins Mrc1 and Tof1 associate with the putative MCM-Cdc45 helicase and limit progression of the replisome when nucleotides are depleted, and the checkpoint kinases Mec1 and Rad53 stabilize such stalled forks and prevent disassembly of the replisome. Forks also pause transiently during unperturbed chromosome replication, at sites where nonnucleosomal proteins bind DNA tightly. We describe a method for inducing prolonged pausing of forks at protein barriers assembled at unique sites on a yeast chromosome, allowing us to examine for the first time the effects of pausing upon replisome integrity. We show that paused forks maintain an intact replisome that contains Mrc1, Tof1, MCM-Cdc45, GINS, and DNA polymerases alpha and epsilon and that recruits the Rrm3 helicase. Surprisingly, pausing does not require Mrc1, although Tof1 and Csm3 are both important. In addition, the integrity of the paused forks does not require Mec1, Rad53, or recombination. We also show that paused forks at analogous barriers in the rDNA are regulated similarly. These data indicate that paused and stalled eukaryotic replisomes resemble each other but are regulated differently.
    • Mrc1 and Tof1 regulate DNA replication forks in different ways during normal S phase.

      Hodgson, Ben; Calzada, Arturo; Labib, Karim; Cancer Research U.K., Paterson Institute for Cancer Research, University of Manchester, Manchester M20 4BX, United Kingdom. (2007-10)
      The Mrc1 and Tof1 proteins are conserved throughout evolution, and in budding yeast they are known to associate with the MCM helicase and regulate the progression of DNA replication forks. Previous work has shown that Mrc1 is important for the activation of checkpoint kinases in responses to defects in S phase, but both Mrc1 and Tof1 also regulate the normal process of chromosome replication. Here, we show that these two important factors control the normal progression of DNA replication forks in distinct ways. The rate of progression of DNA replication forks is greatly reduced in the absence of Mrc1 but much less affected by loss of Tof1. In contrast, Tof1 is critical for DNA replication forks to pause at diverse chromosomal sites where nonnucleosomal proteins bind very tightly to DNA, and this role is not shared with Mrc1.
    • Rapid depletion of budding yeast proteins by fusion to a heat-inducible degron.

      Sanchez-Diaz, Alberto; Kanemaki, Masato; Marchesi, Vanessa; Labib, Karim; Cancer Research U.K., Paterson Institute for Cancer Research, Christie Hospital, Manchester, UK. (2004-03-09)
      One effective way to study the biological function of a protein in vivo is to inactivate it and see what happens to the cell. For proteins that are dispensable for cell viability, the corresponding gene can simply be deleted from its chromosomal locus. The study of essential proteins is more challenging, however, because the function of the protein must be inactivated conditionally. Here, we describe a method that allows the target protein to be depleted rapidly and conditionally, so that the immediate effects on the cell can be examined. The chromosomal locus of a budding yeast gene is modified so that a "heat-inducible degron cassette" is added to the N terminus of the encoded protein, causing it to be degraded by a specific ubiquitin-mediated pathway when cells are shifted from 24 degrees to 37 degrees C. Degradation requires recognition of the degron cassette by the evolutionarily conserved Ubr1 protein, which is associated with a ubiquitin-conjugating enzyme. To promote rapid and conditional depletion of the target protein, we use a yeast strain in which expression of the UBR1 gene can be either repressed or strongly induced. Degron strains are constructed by a simple "one-step" approach using the polymerase chain reaction.