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Yukio Ishimi - One of the best experts on this subject based on the ideXlab platform.

  • function of the amino terminal region of human MCM4 in helicase activity
    Journal of Biochemistry, 2018
    Co-Authors: Xuan Wang, Yukio Ishimi
    Abstract:

    The amino-terminal region of eukaryotic MCM4 is characteristic of the presence of a number of phosphorylation sites for CDK and DDK, suggesting that the region plays regulatory roles in the MCM2-7 helicase function. However, the roles are not fully understood. We analyzed the role of the amino-terminal region of human MCM4 by using MCM4/6/7 helicase as a model for MCM2-7 helicase. First we found that deletion of 35 amino acids at the amino-terminal end resulted in inhibition of DNA helicase activity of the MCM4/6/7 complex. Conversion of arginine at amino acid no. 10 and 11 to alanine had similar effect to the deletion mutant of Δ1-35, suggesting that these arginine play a role in the DNA helicase activity. The data suggest that expression of these mutant MCM4 in HeLa cells perturbed the progression of the S phase. Substitution of six CDK phosphorylation sites (3, 7, 19, 32, 54 and 110) in the amino-terminal region by phospho-mimetic glutamic acids affected the hexamer formation of the MCM4/6/7 complex. MCM4 phosphorylation by CDK may play a role in DNA replication licensing system, and the present results suggest that the phosphorylation interferes MCM function by lowering stability of MCM complex.

  • an MCM4 mutation detected in cancer cells affects MCM4 6 7 complex formation
    Journal of Biochemistry, 2016
    Co-Authors: Ruriko Tatsumi, Yukio Ishimi
    Abstract:

    An MCM4 mutation detected in human cancer cells from endometrium was characterized. The mutation of G486D is located within MCM-box and the glycine at 486 in human MCM4 is conserved in Saccharomyces cerevisiae MCM4 and Sulfolobus solfataricus MCM. This MCM4 mutation affected human MCM4/6/7 complex formation, since the complex containing the mutant MCM4 protein is unstable and the mutant MCM4 protein is tend to be degraded. It is likely that the MCM4 mutation affects the interaction with MCM7 to destabilize the MCM4/6/7 complex. Cells with abnormal nuclear morphology were detected when the mutant MCM4 was expressed in HeLa cells, suggesting that DNA replication was perturbed in the presence of the mutant MCM4. Role of the conserved amino acid in MCM4 function is discussed.

  • g364r mutation of MCM4 detected in human skin cancer cells affects dna helicase activity of MCM4 6 7 complex
    Journal of Biochemistry, 2015
    Co-Authors: Yukio Ishimi, Daiki Irie
    Abstract:

    A number of gene mutations are detected in cells derived from human cancer tissues, but roles of these mutations in cancer cell development are largely unknown. We examined G364R mutation of MCM4 detected in human skin cancer cells. Formation of MCM4/6/7 complex is not affected by the mutation. Consistent with this notion, the binding to MCM6 is comparable between the mutant MCM4 and wild-type MCM4. Nuclear localization of this mutant MCM4 expressed in HeLa cells supports this conclusion. Purified MCM4/6/7 complex containing the G364R MCM4 exhibited similar levels of single-stranded DNA binding and ATPase activities to the complex containing wild-type MCM4. However, the mutant complex showed only 30-50% of DNA helicase activity of the wild-type complex. When G364R MCM4 was expressed in HeLa cells, it was fractionated into nuclease-sensitive chromatin fraction, similar to wild-type MCM4. These results suggest that this mutation does not affect assembly of MCM2-7 complex on replication origins but it interferes some step at function of MCM2-7 helicase. Thus, this mutation may contribute to cancer cell development by disturbing DNA replication.

  • interaction of human minichromosome maintenance protein binding protein with minichromosome maintenance 2 7
    FEBS Journal, 2014
    Co-Authors: Shunsuke Kusunoki, Yukio Ishimi
    Abstract:

    It has been reported that minichromosome maintenance protein-binding protein (MCM-BP) functions in the formation of the pre-replication complex, unloading of minichromosome maintenance (MCM)2-7 from chromatin in late S phase, and formation of the cohesion complex by interacting with MCM3-7 proteins, suggesting that MCM-BP functions in several different reactions during the cell cycle. Here, we examined the interaction of human MCM-BP with MCM2-7 and structural maintenance of chromosome 3 in synchronized HeLa cells by immunoprecipitation. The results show that MCM-BP mainly interacts with MCM7 in the Triton-soluble fraction from S phase and G(2) phase cells, and it also interacts with structural maintenance of chromosome 3 in the fraction from G(2) phase cells. In vitro studies show that MCM-BP disassembles MCM2-7 bound to DNA with a fork-like structure by interacting with MCM3, MCM5, and MCM7. These results suggest that MCM-BP functions in disassembling MCM2-7 on chromatin during S phase and G2 phase by interacting with MCM3, MCM5, and MCM7.

  • effect of an MCM4 mutation that causes tumours in mouse on human MCM4 6 7 complex formation
    Journal of Biochemistry, 2012
    Co-Authors: Emi Watanabe, Rie Ohara, Yukio Ishimi
    Abstract:

    It has been reported that a point mutation of minichromosome maintenance (MCM)4 causes mammary carcinoma, and it deregulates DNA replication to produce abnormal chromosome structures. To understand the effect of this mutation at level of MCM2-7 interaction, we examined the effect of the same mutation of human MCM4 on the complex formation with MCM6 and MCM7 in insect cells. Human MCM4/6/7 complexes containing the mutated MCM4 were formed, but the hexameric complex formation was not evident in comparison with those containing wild-type MCM4. In binary expression of MCM4 and MCM6, decreased levels of MCM6 were recovered with the mutated MCM4, compared with wild-type MCM4. These results suggest that this mutation of MCM4 perturbs proper interaction with MCM6 to affect complex formation of MCM4/6/7 that is a core structure of MCM2-7 complex. Consistent with this notion, nuclear localization and MCM complex formation of forcedly expressed MCM4 in human cells are affected by this mutation. Thus, the defect of this mutant MCM4 in interacting with MCM6 may generate a decreased level of chromatin binding of MCM2-7 complex.

John F. X. Diffley - One of the best experts on this subject based on the ideXlab platform.

  • phosphopeptide binding by sld3 links dbf4 dependent kinase to mcm replicative helicase activation
    The EMBO Journal, 2016
    Co-Authors: Tom D Deegan, Joseph T P Yeeles, John F. X. Diffley
    Abstract:

    Abstract The initiation of eukaryotic DNA replication requires the assembly of active CMG (Cdc45‐MCM‐GINS) helicases at replication origins by a set of conserved and essential firing factors. This process is controlled during the cell cycle by cyclin‐dependent kinase (CDK) and Dbf4‐dependent kinase (DDK), and in response to DNA damage by the checkpoint kinase Rad53/Chk1. Here we show that Sld3, previously shown to be an essential CDK and Rad53 substrate, is recruited to the inactive MCM double hexamer in a DDK‐dependent manner. Sld3 binds specifically to DDK‐phosphorylated peptides from two MCM subunits (MCM4, 6) and then recruits Cdc45. MCM mutants that cannot bind Sld3 or Sld3 mutants that cannot bind phospho‐MCM or Cdc45 do not support replication. Moreover, phosphomimicking mutants in MCM4 and Mcm6 bind Sld3 without DDK and facilitate DDK‐independent replication. Thus, Sld3 is an essential “reader” of DDK phosphorylation, integrating signals from three distinct protein kinase pathways to coordinate DNA replication during S phase.

  • atpase dependent quality control of dna replication origin licensing
    Nature, 2013
    Co-Authors: Jordi Frigola, Dirk Remus, Amina Mehanna, John F. X. Diffley
    Abstract:

    The regulated loading of the Mcm2–7 DNA helicase (comprising six related subunits, Mcm2 to Mcm7) into pre-replicative complexes at multiple replication origins ensures precise once per cell cycle replication in eukaryotic cells. The origin recognition complex (ORC), Cdc6 and Cdt1 load Mcm2–7 into a double hexamer bound around duplex DNA in an ATP-dependent reaction, but the molecular mechanism of this origin ‘licensing’ is still poorly understood. Here we show that both Mcm2–7 hexamers in Saccharomyces cerevisiae are recruited to origins by an essential, conserved carboxy-terminal domain of Mcm3 that interacts with and stimulates the ATPase activity of ORC–Cdc6. ATP hydrolysis can promote Mcm2–7 loading, but can also promote Mcm2–7 release if components are missing or if ORC has been inactivated by cyclin-dependent kinase phosphorylation. Our work provides new insights into how origins are licensed and reveals a novel ATPase-dependent mechanism contributing to precise once per cell cycle replication. The authors describe how the eukaryotic replicative helicase is recruited to origins and reveal a novel ATPase-dependent quality control mechanism. Eukaryotes strictly regulate DNA replication so that it occurs once per cell cycle. This condition is maintained by a 'licensing' process, involving the ORC, Cdc6, Cdt1 and Mcm2–7 proteins. The stability of this process is essential for stable inheritance of large genomes in the eukaryotic cell cycle. In this study, John Diffley and colleagues show that in budding yeast, ORC–Cdc6 recruits the Mcm2–7 double hexamer through the Mcm3 subunit. The interaction stimulates the ATPase activity of ORC–Cdc6. However, this hydrolysis can promote either Mcm loading or its release, depending on whether the pre-replication complex is complete and the stage of the cell cycle is appropriate for activation of the origin. This kinetic proofreading mechanism prevents the accumulation of partially assembled, dead-end complexes.

  • atpase dependent quality control of dna replication origin licensing
    Nature, 2013
    Co-Authors: Jordi Frigola, Dirk Remus, Amina Mehanna, John F. X. Diffley
    Abstract:

    The regulated loading of the Mcm2-7 DNA helicase (comprising six related subunits, Mcm2 to Mcm7) into pre-replicative complexes at multiple replication origins ensures precise once per cell cycle replication in eukaryotic cells. The origin recognition complex (ORC), Cdc6 and Cdt1 load Mcm2-7 into a double hexamer bound around duplex DNA in an ATP-dependent reaction, but the molecular mechanism of this origin 'licensing' is still poorly understood. Here we show that both Mcm2-7 hexamers in Saccharomyces cerevisiae are recruited to origins by an essential, conserved carboxy-terminal domain of Mcm3 that interacts with and stimulates the ATPase activity of ORC-Cdc6. ATP hydrolysis can promote Mcm2-7 loading, but can also promote Mcm2-7 release if components are missing or if ORC has been inactivated by cyclin-dependent kinase phosphorylation. Our work provides new insights into how origins are licensed and reveals a novel ATPase-dependent mechanism contributing to precise once per cell cycle replication.

Daniel L. Kaplan - One of the best experts on this subject based on the ideXlab platform.

  • a positive amplification mechanism involving a kinase and replication initiation factor helps assemble the replication fork helicase
    Journal of Biological Chemistry, 2017
    Co-Authors: Irina Bruck, Nalini Dhingra, Daniel L. Kaplan
    Abstract:

    : The assembly of the replication fork helicase during S phase is key to the initiation of DNA replication in eukaryotic cells. One step in this assembly in budding yeast is the association of Cdc45 with the Mcm2-7 heterohexameric ATPase, and a second step is the assembly of the tetrameric GINS (GG-Ichi-Nii-San) complex with Mcm2-7. Dbf4-dependent kinase (DDK) and S-phase cyclin-dependent kinase (S-CDK) are two S phase-specific kinases that phosphorylate replication proteins during S phase, and Dpb11, Sld2, Sld3, Pol ϵ, and Mcm10 are factors that are also required for replication initiation. However, the exact roles of these initiation factors in assembly of the replication fork helicase remain unclear. We show here that Dpb11 stimulates DDK phosphorylation of the minichromosome maintenance complex protein MCM4 alone and also of the Mcm2-7 complex and the dsDNA-loaded Mcm2-7 complex. We further demonstrate that Dpb11 can directly recruit DDK to MCM4. A DDK phosphomimetic mutant of MCM4 bound Dpb11 with substantially higher affinity than wild-type MCM4, suggesting a mechanism to recruit Dpb11 to DDK-phosphorylated Mcm2-7. Furthermore, dsDNA-loaded Mcm2-7 harboring the DDK phosphomimetic MCM4 mutant bound GINS in the presence of Dpb11, suggesting a mechanism for how GINS is recruited to Mcm2-7. We isolated a mutant of Dpb11 that is specifically defective for binding to MCM4. This mutant, when expressed in budding yeast, diminished cell growth and DNA replication, substantially decreased MCM4 phosphorylation, and decreased association of GINS with replication origins. We conclude that Dpb11 functions with DDK and MCM4 in a positive amplification mechanism to trigger the assembly of the replication fork helicase.

  • the replication initiation protein sld3 treslin orchestrates the assembly of the replication fork helicase during s phase
    Journal of Biological Chemistry, 2015
    Co-Authors: Irina Bruck, Daniel L. Kaplan
    Abstract:

    The initiation of DNA replication is a highly regulated process in eukaryotic cells, and central to the process of initiation is the assembly and activation of the replication fork helicase. The replication fork helicase is comprised of CMG (Cdc45, Mcm2-7, and GINS) in eukaryotic cells, and the mechanism underlying assembly of the CMG during S phase was studied in this article. We identified a point mutation of Sld3 that is specifically defective for Mcm3 and Mcm5 interaction (sld3-m10), and also identified a point mutation of Sld3 that is specifically defective for single-stranded DNA (ssDNA) interaction (sld3-m9). Expression of wild-type levels of sld3-m9 resulted in a severe DNA replication defect with no recruitment of GINS to Mcm2-7, whereas expression of wild-type levels of sld3-m10 resulted in a severe replication defect with no Cdc45 recruitment to Mcm2-7. We propose a model for Sld3-mediated control of replication initiation, wherein Sld3 manages the proper assembly of the CMG during S phase. We also find that the biochemical functions identified for Sld3 are conserved in human Treslin, suggesting that Treslin orchestrates assembly of the CMG in human cells.

  • the dbf4 cdc7 kinase promotes mcm2 7 ring opening to allow for single stranded dna extrusion and helicase assembly
    Journal of Biological Chemistry, 2015
    Co-Authors: Irina Bruck, Daniel L. Kaplan
    Abstract:

    The replication fork helicase in eukaryotes is composed of Cdc45, Mcm2-7, and GINS (CMG). The Dbf4-Cdc7 kinase phosphorylates Mcm2 in vitro, but the in vivo role for Dbf4-Cdc7 phosphorylation of Mcm2 is unclear. We find that budding yeast Dbf4-Cdc7 phosphorylates Mcm2 in vivo under normal conditions during S phase. Inhibiting Dbf4-Cdc7 phosphorylation of Mcm2 confers a dominant-negative phenotype with a severe growth defect. Inhibiting Dbf4-Cdc7 phosphorylation of Mcm2 under wild-type expression conditions also results in impaired DNA replication, substantially decreased single-stranded formation at an origin, and markedly disrupted interaction between GINS and Mcm2-7 during S phase. In vitro, Dbf4-Cdc7 kinase (DDK) phosphorylation of Mcm2 substantially weakens the interaction between Mcm2 and Mcm5, and Dbf4-Cdc7 phosphorylation of Mcm2 promotes Mcm2-7 ring opening. The extrusion of ssDNA from the central channel of Mcm2-7 triggers GINS attachment to Mcm2-7. Thus, Dbf4-Cdc7 phosphorylation of Mcm2 may open the Mcm2-7 ring at the Mcm2-Mcm5 interface, allowing for single-stranded DNA extrusion and subsequent GINS assembly with Mcm2-7.

Hisao Masai - One of the best experts on this subject based on the ideXlab platform.

  • cdt1 forms a complex with the minichromosome maintenance protein mcm and activates its helicase activity
    Journal of Biological Chemistry, 2008
    Co-Authors: Hisao Masai
    Abstract:

    MCM4/6/7 forms a complex possessing DNA helicase activity, suggesting that Mcm may be a central component for the replicative helicase. Although Cdt1 is known to be essential for loading of Mcm onto the chromatin, its precise role in pre-RC formation and replication initiation is unknown. Using purified proteins, we show that Cdt1 forms a complex with MCM4/6/7, Mcm2/3/4/5/6/7, and Mcm2/4/6/7 in glycerol gradient fractionation through interaction with Mcm2 and MCM4/6. In the glycerol gradient fractionation, MCM4/6/7-Cdt1 forms a complex (speculated to be a (MCM4/6/7)2-Cdt13 assembly) in the presence of ATP, which is significantly larger than the MCM4/6/7-Cdt1 complex generated in its absence. Furthermore, DNA binding and helicase activities of MCM4/6/7 are significantly stimulated by Cdt1 protein in vitro. We generd a Cdt1 mutant, which fails to stimulate DNA binding and helicase activities of4/6/7. This mutant Cdt1 showed reduced interaction with Mcm and is deficient in the formation of a high molecular weight complex with Mcm. Thus, a productive interaction between Cdt1 and MCM appears to be essential for efficient loading of MCM onto template DNA, as well as for the efficient unwinding reaction.

  • phosphorylation of MCM4 by cdc7 kinase facilitates its interaction with cdc45 on the chromatin
    Journal of Biological Chemistry, 2006
    Co-Authors: Hisao Masai, Etsuko Matsui, Chika Taniyama, Keiko Ogino, Naoko Kakusho, Seiji Matsumoto, Jung Min Kim, Ai Ishii, Taku Tanaka, Toshiko Kobayashi
    Abstract:

    Cdc7 kinase, conserved from yeasts to human, plays important roles in DNA replication. However, the mechanisms by which it stimulates initiation of DNA replication remain largely unclear. We have analyzed phosphorylation of MCM subunits during cell cycle by examining mobility shift on SDS-PAGE. MCM4 on the chromatin undergoes specific phosphorylation during S phase. Cdc7 phosphorylates MCM4 in the MCM complexes as well as the MCM4 N-terminal polypeptide. Experiments with phospho-amino acid-specific antibodies indicate that the S phase-specific mobility shift is due to the phosphorylation at specific N-terminal (S/T)(S/T)P residues of the MCM4 protein. These specific phosphorylation events are not observed in mouse ES cells deficient in Cdc7 or are reduced in the cells treated with siRNA specific to Cdc7, suggesting that they are mediated by Cdc7 kinase. The N-terminal phosphorylation of MCM4 stimulates association of Cdc45 with the chromatin, suggesting that it may be an important phosphorylation event by Cdc7 for activation of replication origins. Deletion of the N-terminal non-conserved 150 amino acids of MCM4 results in growth inhibition, and addition of amino acids carrying putative Cdc7 target sequences partially restores the growth. Furthermore, combination of MCM4 N-terminal deletion with alanine substitution and deletion of the N-terminal segments of MCM2 and MCM6, respectively, which contain clusters of serine/threonine and are also likely targets of Cdc7, led to an apparent nonviable phenotype. These results are consistent with the notion that the N-terminal phosphorylation of MCM2, MCM4, and MCM6 may play functionally redundant but essential roles in initiation of DNA replication.

  • roles of mcm7 and MCM4 subunits in the dna helicase activity of the mouse MCM4 6 7 complex
    Journal of Biological Chemistry, 2002
    Co-Authors: Zhiying You, Hisao Masai, Yukio Ishimi, Fumio Hanaoka
    Abstract:

    Abstract Mcm, which is composed of six structurally related subunits (Mcm2–7), is essential for eukaryotic DNA replication. A subassembly of Mcm, the MCM4/6/7 double-trimeric complex, possesses DNA helicase activity, and it has been proposed that Mcm may function as a replicative helicase at replication forks. We show here that conserved ATPase motifs of Mcm7 are essential for ATPase and DNA helicase activities of the MCM4/6/7 complex. Because uncomplexed Mcm7 displayed neither ATPase nor DNA helicase activity, Mcm7 contributes to the DNA helicase activity of the Mcm complex through interaction with other subunits. In contrast, the MCM4/6/7 complex containing a zinc finger mutant of MCM4 with partially impaired DNA binding activity exhibited elevated DNA helicase activity. The MCM4/6/7 complex containing this MCM4 mutant tended to dissociate into trimeric complexes, suggesting that the zinc finger of MCM4 is involved in subunit interactions of trimers. The MCM4 mutants lacking the N-terminal 35 or 112 amino acids could form hexameric MCM4/6/7 complexes, but displayed very little DNA helicase activity. In conjunction with the previously reported essential role of Mcm6 in ATP binding (You, Z., Komamura, Y., and Ishimi, Y. (1999) Mol. Cell. Biol. 19, 8003–8015), our data indicate distinct roles of MCM4, Mcm6, and Mcm7 subunits in activation of the DNA helicase activity of the MCM4/6/7 complex.

  • human cdc7 related kinase complex in vitro phosphorylation of mcm by concerted actions of cdks and cdc7 and that of a criticial threonine residue of cdc7 by cdks
    Journal of Biological Chemistry, 2000
    Co-Authors: Hisao Masai, Zhiying You, Yukio Ishimi, Etsuko Matsui, Katsuyuki Tamai, Kenichi Arai
    Abstract:

    Abstract huCdc7 encodes a catalytic subunit forSaccharomyces cerevisae Cdc7-related kinase complex of human. ASK, whose expression is cell cycle-regulated, binds and activates huCdc7 kinase in a cell cycle-dependent manner (Kumagai, H., Sato, N., Yamada, M., Mahony, D., Seghezzi, W., Lees, E., Arai, K., and Masai, H. (1999) Mol. Cell. Biol. 19, 5083–5095). We have expressed huCdc7 complexed with ASK regulatory subunit using the insect cell expression system. To facilitate purification of the kinase complex, glutathioneS-transferase (GST) was fused to huCdc7 and GST-huCdc7-ASK complex was purified. GST-huCdc7 protein is inert as a kinase on its own, and phosphorylation absolutely depends on the presence of the ASK subunit. It autophosphorylates both subunits in vitro and phosphorylates a number of replication proteins to different extents. Among them, MCM2 protein, either in a free form or in a MCM2-4-6-7 complex, serves as an excellent substrate for huCdc7-ASK kinase complexin vitro. MCM4 and MCM6 are also phosphorylated by huCdc7 albeit to less extent. MCM2 and -4 in the MCM2-4-6-7 complex are phosphorylated by Cdks as well, and prior phosphorylation of the MCM2-4-6-7 complex by Cdks facilitates phosphorylation of MCM2 by huCdc7, suggesting collaboration between Cdks and Cdc7 in phosphorylation of MCM for initiation of S phase. huCdc7 and ASK proteins can also be phosphorylated by Cdks in vitro. Among four possible Cdk phosphorylation sites of huCdc7, replacement of Thr-376, corresponding to the activating threonine of Cdk, with alanine (T376A mutant) dramatically reduces kinase activity, indicative of kinase activation by phosphorylation of this residue. In vitro, Cdk2-Cyclin E, Cdk2-Cyclin A, and Cdc2-Cyclin B, but not Cdk4-Cyclin D1, phosphorylates the Thr-376 residue of huCdc7, suggesting possible regulation of huCdc7 by Cdks.

Zhiying You - One of the best experts on this subject based on the ideXlab platform.

  • roles of mcm7 and MCM4 subunits in the dna helicase activity of the mouse MCM4 6 7 complex
    Journal of Biological Chemistry, 2002
    Co-Authors: Zhiying You, Hisao Masai, Yukio Ishimi, Fumio Hanaoka
    Abstract:

    Abstract Mcm, which is composed of six structurally related subunits (Mcm2–7), is essential for eukaryotic DNA replication. A subassembly of Mcm, the MCM4/6/7 double-trimeric complex, possesses DNA helicase activity, and it has been proposed that Mcm may function as a replicative helicase at replication forks. We show here that conserved ATPase motifs of Mcm7 are essential for ATPase and DNA helicase activities of the MCM4/6/7 complex. Because uncomplexed Mcm7 displayed neither ATPase nor DNA helicase activity, Mcm7 contributes to the DNA helicase activity of the Mcm complex through interaction with other subunits. In contrast, the MCM4/6/7 complex containing a zinc finger mutant of MCM4 with partially impaired DNA binding activity exhibited elevated DNA helicase activity. The MCM4/6/7 complex containing this MCM4 mutant tended to dissociate into trimeric complexes, suggesting that the zinc finger of MCM4 is involved in subunit interactions of trimers. The MCM4 mutants lacking the N-terminal 35 or 112 amino acids could form hexameric MCM4/6/7 complexes, but displayed very little DNA helicase activity. In conjunction with the previously reported essential role of Mcm6 in ATP binding (You, Z., Komamura, Y., and Ishimi, Y. (1999) Mol. Cell. Biol. 19, 8003–8015), our data indicate distinct roles of MCM4, Mcm6, and Mcm7 subunits in activation of the DNA helicase activity of the MCM4/6/7 complex.

  • human cdc7 related kinase complex in vitro phosphorylation of mcm by concerted actions of cdks and cdc7 and that of a criticial threonine residue of cdc7 by cdks
    Journal of Biological Chemistry, 2000
    Co-Authors: Hisao Masai, Zhiying You, Yukio Ishimi, Etsuko Matsui, Katsuyuki Tamai, Kenichi Arai
    Abstract:

    Abstract huCdc7 encodes a catalytic subunit forSaccharomyces cerevisae Cdc7-related kinase complex of human. ASK, whose expression is cell cycle-regulated, binds and activates huCdc7 kinase in a cell cycle-dependent manner (Kumagai, H., Sato, N., Yamada, M., Mahony, D., Seghezzi, W., Lees, E., Arai, K., and Masai, H. (1999) Mol. Cell. Biol. 19, 5083–5095). We have expressed huCdc7 complexed with ASK regulatory subunit using the insect cell expression system. To facilitate purification of the kinase complex, glutathioneS-transferase (GST) was fused to huCdc7 and GST-huCdc7-ASK complex was purified. GST-huCdc7 protein is inert as a kinase on its own, and phosphorylation absolutely depends on the presence of the ASK subunit. It autophosphorylates both subunits in vitro and phosphorylates a number of replication proteins to different extents. Among them, MCM2 protein, either in a free form or in a MCM2-4-6-7 complex, serves as an excellent substrate for huCdc7-ASK kinase complexin vitro. MCM4 and MCM6 are also phosphorylated by huCdc7 albeit to less extent. MCM2 and -4 in the MCM2-4-6-7 complex are phosphorylated by Cdks as well, and prior phosphorylation of the MCM2-4-6-7 complex by Cdks facilitates phosphorylation of MCM2 by huCdc7, suggesting collaboration between Cdks and Cdc7 in phosphorylation of MCM for initiation of S phase. huCdc7 and ASK proteins can also be phosphorylated by Cdks in vitro. Among four possible Cdk phosphorylation sites of huCdc7, replacement of Thr-376, corresponding to the activating threonine of Cdk, with alanine (T376A mutant) dramatically reduces kinase activity, indicative of kinase activation by phosphorylation of this residue. In vitro, Cdk2-Cyclin E, Cdk2-Cyclin A, and Cdc2-Cyclin B, but not Cdk4-Cyclin D1, phosphorylates the Thr-376 residue of huCdc7, suggesting possible regulation of huCdc7 by Cdks.

  • Biochemical Analysis of the Intrinsic MCM4-Mcm6-Mcm7 DNA Helicase Activity
    Molecular and Cellular Biology, 1999
    Co-Authors: Zhiying You, Yuki Komamura, Yukio Ishimi
    Abstract:

    Mcm proteins play an essential role in eukaryotic DNA replication, but their biochemical functions are poorly understood. Recently, we reported that a DNA helicase activity is associated with an MCM4-Mcm6-Mcm7 (MCM4,6,7) complex, suggesting that this complex is involved in the initiation of DNA replication as a DNA-unwinding enzyme. In this study, we have expressed and isolated the mouse Mcm2,4,6,7 proteins from insect cells and characterized various mutant MCM4,6,7 complexes in which the conserved ATPase motifs of the MCM4 and Mcm6 proteins were mutated. The activities associated with such preparations demonstrated that the DNA helicase activity is intrinsically associated with the MCM4,6,7 complex. Biochemical analyses of these mutant MCM4,6,7 complexes indicated that the ATP binding activity of the Mcm6 protein in the complex is critical for DNA helicase activity and that the MCM4 protein may play a role in the single-stranded DNA binding activity of the complex. The results also indicated that the two activities of DNA helicase and single-stranded DNA binding can be separated.