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E. Schwob - One of the best experts on this subject based on the ideXlab platform.

  • identification of tah11 sid2 as the ortholog of the replication Licensing Factor cdt1 in saccharomyces cerevisiae
    Current Biology, 2002
    Co-Authors: A. Devault, E. A. Vallen, T. Yuan, S. Green, A. Bensimon, E. Schwob
    Abstract:

    Summary in origin Licensing are sensitive to lesions or damage generated by this topoisomerase allele, but it is worth Faithful duplication of the genetic material requires noting that two other genes, CDC45 and DPB11 , re- that replication origins fire only once per cell cycle. quired for the initiation of DNA replication or an early Centraltothiscontrolisthetightlyregulatedformation step in elongation were also recovered [10]. An allele of of prereplicative complexes (preRCs) at future origins YJR046w wasindependently isolatedas syntheticlethal of DNA replication [1]. In all eukaryotes studied, this with a deletion of the Cdk inhibitor SIC1 , hence its sec- entails loading by Cdc6 of the Mcm2-7 helicase next ond name, SID2 (Sic1 Indispensable) [9]. In that work, to the origin recognition complex (ORC) [2]. More re- two mutations were characterized; one ( sid2-1 ) that cently, another Factor, named Cdt1, was shown to be causes a severe growth defect in conjunction with sic1 essentialforMcmloadinginfissionyeastand

  • Identification of Tah11/Sid2 as the Ortholog of the Replication Licensing Factor Cdt1 in Saccharomyces cerevisiae
    Current biology : CB, 2002
    Co-Authors: A. Devault, E. A. Vallen, T. Yuan, S. Green, A. Bensimon, E. Schwob
    Abstract:

    Summary in origin Licensing are sensitive to lesions or damage generated by this topoisomerase allele, but it is worth Faithful duplication of the genetic material requires noting that two other genes, CDC45 and DPB11 , re- that replication origins fire only once per cell cycle. quired for the initiation of DNA replication or an early Centraltothiscontrolisthetightlyregulatedformation step in elongation were also recovered [10]. An allele of of prereplicative complexes (preRCs) at future origins YJR046w wasindependently isolatedas syntheticlethal of DNA replication [1]. In all eukaryotes studied, this with a deletion of the Cdk inhibitor SIC1 , hence its sec- entails loading by Cdc6 of the Mcm2-7 helicase next ond name, SID2 (Sic1 Indispensable) [9]. In that work, to the origin recognition complex (ORC) [2]. More re- two mutations were characterized; one ( sid2-1 ) that cently, another Factor, named Cdt1, was shown to be causes a severe growth defect in conjunction with sic1 essentialforMcmloadinginfissionyeastand

  • Identification of Tah11/Sid2 as the ortholog of the replication Licensing Factor Cdt1 in Saccharomyces cerevisiae
    Current Biology - CB, 2002
    Co-Authors: A. Devault, E. A. Vallen, T. Yuan, S. Green, A. Bensimon, E. Schwob
    Abstract:

    Faithful duplication of the genetic material requires that replication origins fire only once per cell cycle. Central to this control is the tightly regulated formation of prereplicative complexes (preRCs) at future origins of DNA replication. In all eukaryotes studied, this entails loading by Cdc6 of the Mcm2-7 helicase next to the origin recognition complex (ORC). More recently, another Factor, named Cdt1, was shown to be essential for Mcm loading in fission yeast and Xenopus as well as for DNA replication in Drosophila and humans. Surprisingly, no Cdt1 homolog was found in budding yeast, despite the conserved nature of origin Licensing. Here we identify Tah11/Sid2, previously isolated through interactions with topoisomerase and Cdk inhibitor mutants, as an ortholog of Cdt1. We show that sid2 mutants lose minichromosomes in an ARS number-dependent manner, consistent with ScCdt1/Sid2 being involved in origin Licensing. Accordingly, cells partially depleted of Cdt1 replicate DNA from fewer origins, whereas fully depleted cells fail to load Mcm2 on chromatin and fail to initiate but not elongate DNA synthesis. We conclude that origin Licensing depends in S. cerevisiae as in other eukaryotes on both Cdc6 and Cdt1.

Hideo Nishitani - One of the best experts on this subject based on the ideXlab platform.

  • Mismatch repair proteins recruited to ultraviolet light-damaged sites lead to degradation of Licensing Factor Cdt1 in the G1 phase.
    Cell cycle (Georgetown Tex.), 2017
    Co-Authors: Miyuki Tanaka, Yasushi Shiomi, Akiyo Hayashi, Michiyo Takahara, Kohei Nukina, Wataru Sakai, Kaoru Sugasawa, Hideo Nishitani
    Abstract:

    Cdt1 is rapidly degraded by CRL4Cdt2 E3 ubiquitin ligase after UV (UV) irradiation. Previous reports revealed that the nucleotide excision repair (NER) pathway is responsible for the rapid Cdt1-proteolysis. Here, we show that mismatch repair (MMR) proteins are also involved in the degradation of Cdt1 after UV irradiation in the G1 phase. First, compared with the rapid (within ∼15 min) degradation of Cdt1 in normal fibroblasts, Cdt1 remained stable for ∼30 min in NER-deficient XP-A cells, but was degraded within ∼60 min. The delayed degradation was also dependent on PCNA and CRL4Cdt2. The MMR proteins Msh2 and Msh6 were recruited to the UV-damaged sites of XP-A cells in the G1 phase. Depletion of these Factors with small interfering RNAs prevented Cdt1 degradation in XP-A cells. Similar to the findings in XP-A cells, depletion of XPA delayed Cdt1 degradation in normal fibroblasts and U2OS cells, and co-depletion of Msh6 further prevented Cdt1 degradation. Furthermore, depletion of Msh6 alone delayed Cdt1 degradation in both cell types. When Cdt1 degradation was attenuated by high Cdt1 expression, repair synthesis at the damaged sites was inhibited. Our findings demonstrate that UV irradiation induces multiple repair pathways that activate CRL4Cdt2 to degrade its target proteins in the G1 phase of the cell cycle, leading to efficient repair of DNA damage.

  • Imaging Analysis to Determine Chromatin Binding of the Licensing Factor MCM2-7 in Mammalian Cells
    Methods in molecular biology (Clifton N.J.), 2014
    Co-Authors: Masayuki Morino, Yasushi Shiomi, Miyuki Tanaka, Hideo Nishitani
    Abstract:

    S-CDK and DDK protein kinases initiate DNA replication at replication origins. Prior to the activation of these kinases, origins must become competent for replication by loading MCM2-7 DNA helicase on chromatin. This process is known as replication Licensing or pre-replicative complex (pre-RC) formation. After the onset of S phase, however, Licensing is inhibited to prevent re-replication of DNA. In this chapter, we describe a method to analyze origin Licensing by imaging the chromatin-bound Licensing Factor MCM2-7. In a normal cell cycle, MCM2-7 is loaded at the end of mitosis or early G1 phase. As S phase progresses, MCM2-7 is dissociated from the replicated regions. When DNA replication is completed, cells in G2 phase have no chromatin-bound MCM2-7. The analysis of chromatin-bound MCM2-7 in each cell provides an insight into cell cycle stage and condition for cell cycle.

  • Imaging Analysis of Cell Cycle-Dependent Degradation of Cdt1 in Mammalian Cells
    Methods in molecular biology (Clifton N.J.), 2014
    Co-Authors: Yasushi Shiomi, Miyuki Tanaka, Naohiro Suenaga, Akiyo Hayashi, Hideo Nishitani
    Abstract:

    Numerous cell cycle-regulating proteins are controlled by protein degradation. Recent work shows that ubiquitination-dependent proteolysis plays an important role in once-per-cell cycle control of DNA replication. Cdt1 is a Licensing Factor essential for assembling the pre-replicative complex on replication origins. Cdt1 is present in G1 phase, but after S phase ubiquitin-mediated proteolysis maintains Cdt1 at low levels. This is important to prevent the re-replication of chromosomal DNA. The cell cycle-dependent degradation of Cdt1 can be monitored by dual staining of the cell nuclei with antibodies against Cdt1- and S/G2-phase marker proteins, such as cyclin A or geminin.

  • Dynamic recruitment of Licensing Factor Cdt1 to sites of DNA damage.
    Journal of cell science, 2011
    Co-Authors: Vassilis Roukos, Hideo Nishitani, Ali Kinkhabwala, Julien Colombelli, Panagiotis Kotsantis, Stavros Taraviras, Ernst Stelzer, Philippe Bastiaens, Zoi Lygerou
    Abstract:

    For genomic integrity to be maintained, the cell cycle and DNA damage responses must be linked. Cdt1, a G1-specific cell-cycle Factor, is targeted for proteolysis by the Cul4-Ddb1(Cdt2) ubiquitin ligase following DNA damage. Using a laser nanosurgery microscope to generate spatially restricted DNA damage within the living cell nucleus, we show that Cdt1 is recruited onto damaged sites in G1 phase cells, within seconds of DNA damage induction. PCNA, Cdt2, Cul4, DDB1 and p21(Cip1) also accumulate rapidly to damaged sites. Cdt1 recruitment is PCNA-dependent, whereas PCNA and Cdt2 recruitment are independent of Cdt1. Fitting of fluorescence recovery after photobleaching profiles to an analytic reaction-diffusion model shows that Cdt1 and p21(Cip1) exhibit highly dynamic binding at the site of damage, whereas PCNA appears immobile. Cdt2 exhibits both a rapidly exchanging and an apparently immobile subpopulation. Our data suggest that PCNA provides an immobile binding interface for dynamic Cdt1 interactions at the site of damage, which leads to rapid Cdt1 recruitment to damaged DNA, preceding Cdt1 degradation.

  • cdk inhibitor p21 is degraded by a proliferating cell nuclear antigen coupled cul4 ddb1cdt2 pathway during s phase and after uv irradiation
    Journal of Biological Chemistry, 2008
    Co-Authors: Hideo Nishitani, Yasushi Shiomi, Hiroka Iida, Masato Michishita, Toshihiro Takami, Toshiki Tsurimoto
    Abstract:

    Previous reports showed that chromatin-associated PCNA couples DNA replication with Cul4-DDB1Cdt2-dependent proteolysis of the Licensing Factor Cdt1. The CDK inhibitor p21, another PCNA-binding protein, is also degraded both in S phase and after UV irradiation. Here we show that p21 is degraded by the same ubiquitin-proteasome pathway as Cdt1 in HeLa cells. When PCNA or components of Cul4-DDB1Cdt2 were silenced or when the PCNA binding site on p21 was mutated, degradation of p21 was prevented both in S phase and after UV irradiation. p21 was co-immunoprecipitated with Cul4A and DDB1 proteins when expressed in cells. The purified Cul4A-DDB1Cdt2 complex ubiquitinated p21 in vitro. Consistently, p21 protein levels are low during S phase and increase around G2 phase. Mutational analysis suggested that in addition to the PCNA binding domain, its flanking regions are also important for recognition by Cul4-DDB1Cdt2. Our findings provide a new aspect of proteolytic control of p21 during the cell cycle.

Jeanette Gowen Cook - One of the best experts on this subject based on the ideXlab platform.

  • Recruitment of the human Cdt1 replication Licensing protein by the loop domain of Hec1 is required for stable kinetochore microtubule attachment
    Nature cell biology, 2012
    Co-Authors: Dileep Varma, Edward D. Salmon, Jennifer G Deluca, Lynsie J.r. Sundin, Srikripa Chandrasekaran, Karen T. Reidy, Xiaohu Wan, Dawn Chasse, Kathleen R. Nevis, Jeanette Gowen Cook
    Abstract:

    The replication origin Licensing Factor Cdt1 is now demonstrated to function at the kinetochore in mitosis. Cook, Salmon and colleagues show that Cdt1 binds to the loop domain of Hec1 in the Ndc80 kinetochore complex and stabilizes kinetochore–microtubule attachment.

  • Stress-Stimulated Mitogen-Activated Protein Kinases Control the Stability and Activity of the Cdt1 DNA Replication Licensing Factor
    Molecular and cellular biology, 2011
    Co-Authors: Srikripa Chandrasekaran, Ting Xu Tan, Jonathan R. Hall, Jeanette Gowen Cook
    Abstract:

    DNA replication is tightly coordinated both with cell cycle cues and with responses to extracellular signals to maintain genome stability. We discovered that human Cdt1, an essential origin Licensing protein whose activity must be restricted to G(1) phase, is a substrate of the stress-activated mitogen-activated protein (MAP) kinases p38 and c-Jun N-terminal kinase (JNK). These MAP kinases phosphorylate Cdt1 both during unperturbed G(2) phase and during an acute stress response. Phosphorylation renders Cdt1 resistant to ubiquitin-mediated degradation during S phase and after DNA damage by blocking Cdt1 binding to the Cul4 adaptor, Cdt2. Mutations that block normal cell cycle-regulated MAP kinase-mediated phosphorylation interfere with rapid Cdt1 reaccumulation at the end of S phase. Phosphomimetic mutations recapitulate the stabilizing effects of Cdt1 phosphorylation but also reduce the ability of Cdt1 to support origin Licensing. Two other CRL4(Cdt2) targets, the cyclin-dependent kinase (CDK) inhibitor p21 and the methyltransferase PR-Set7/Set8, are similarly stabilized by MAP kinase activity. These findings support a model in which MAP kinase activity in G(2) promotes reaccumulation of a low-activity Cdt1 isoform after replication is complete.

  • coordinated activation of the origin Licensing Factor cdc6 and cdk2 in resting human fibroblasts expressing sv40 small t antigen and cyclin e
    Journal of Biological Chemistry, 2009
    Co-Authors: Elena Sotillo, Jeanette Gowen Cook, Judit Garriga, Amol Padgaonkar, Alison Kurimchak, Xavier Grana
    Abstract:

    We have previously shown that SV40 small t antigen (st) cooperates with deregulated cyclin E to activate CDK2 and bypass quiescence in normal human fibroblasts (NHF). Here we show that st expression in serum-starved and density-arrested NHF specifically induces up-regulation and loading of CDC6 onto chromatin. Coexpression of cyclin E results in further accumulation of CDC6 onto chromatin concomitantly with phosphorylation of CDK2 on Thr-160 and CDC6 on Ser-54. Investigation of the mechanism leading to CDC6 accumulation and chromatin loading indicates that st is a potent inducer of cdc6 mRNA expression and increases CDC6 protein stability. We also show that CDC6 expression in quiescent NHF efficiently promotes cyclin E loading onto chromatin, but it is not sufficient to activate CDK2. Moreover, we show that CDC6 expression is linked to phosphorylation of the activating T loop of CDK2 in serum-starved NHF stimulated with mitogens or ectopically expressing cyclin E and st. Our data suggest a model where the combination of st and deregulated cyclin E result in cooperative and coordinated activation of both an essential origin Licensing Factor, CDC6, and an activity required for origin firing, CDK2, resulting in progression from quiescence to S phase.

A. Devault - One of the best experts on this subject based on the ideXlab platform.

  • identification of tah11 sid2 as the ortholog of the replication Licensing Factor cdt1 in saccharomyces cerevisiae
    Current Biology, 2002
    Co-Authors: A. Devault, E. A. Vallen, T. Yuan, S. Green, A. Bensimon, E. Schwob
    Abstract:

    Summary in origin Licensing are sensitive to lesions or damage generated by this topoisomerase allele, but it is worth Faithful duplication of the genetic material requires noting that two other genes, CDC45 and DPB11 , re- that replication origins fire only once per cell cycle. quired for the initiation of DNA replication or an early Centraltothiscontrolisthetightlyregulatedformation step in elongation were also recovered [10]. An allele of of prereplicative complexes (preRCs) at future origins YJR046w wasindependently isolatedas syntheticlethal of DNA replication [1]. In all eukaryotes studied, this with a deletion of the Cdk inhibitor SIC1 , hence its sec- entails loading by Cdc6 of the Mcm2-7 helicase next ond name, SID2 (Sic1 Indispensable) [9]. In that work, to the origin recognition complex (ORC) [2]. More re- two mutations were characterized; one ( sid2-1 ) that cently, another Factor, named Cdt1, was shown to be causes a severe growth defect in conjunction with sic1 essentialforMcmloadinginfissionyeastand

  • Identification of Tah11/Sid2 as the Ortholog of the Replication Licensing Factor Cdt1 in Saccharomyces cerevisiae
    Current biology : CB, 2002
    Co-Authors: A. Devault, E. A. Vallen, T. Yuan, S. Green, A. Bensimon, E. Schwob
    Abstract:

    Summary in origin Licensing are sensitive to lesions or damage generated by this topoisomerase allele, but it is worth Faithful duplication of the genetic material requires noting that two other genes, CDC45 and DPB11 , re- that replication origins fire only once per cell cycle. quired for the initiation of DNA replication or an early Centraltothiscontrolisthetightlyregulatedformation step in elongation were also recovered [10]. An allele of of prereplicative complexes (preRCs) at future origins YJR046w wasindependently isolatedas syntheticlethal of DNA replication [1]. In all eukaryotes studied, this with a deletion of the Cdk inhibitor SIC1 , hence its sec- entails loading by Cdc6 of the Mcm2-7 helicase next ond name, SID2 (Sic1 Indispensable) [9]. In that work, to the origin recognition complex (ORC) [2]. More re- two mutations were characterized; one ( sid2-1 ) that cently, another Factor, named Cdt1, was shown to be causes a severe growth defect in conjunction with sic1 essentialforMcmloadinginfissionyeastand

  • Identification of Tah11/Sid2 as the ortholog of the replication Licensing Factor Cdt1 in Saccharomyces cerevisiae
    Current Biology - CB, 2002
    Co-Authors: A. Devault, E. A. Vallen, T. Yuan, S. Green, A. Bensimon, E. Schwob
    Abstract:

    Faithful duplication of the genetic material requires that replication origins fire only once per cell cycle. Central to this control is the tightly regulated formation of prereplicative complexes (preRCs) at future origins of DNA replication. In all eukaryotes studied, this entails loading by Cdc6 of the Mcm2-7 helicase next to the origin recognition complex (ORC). More recently, another Factor, named Cdt1, was shown to be essential for Mcm loading in fission yeast and Xenopus as well as for DNA replication in Drosophila and humans. Surprisingly, no Cdt1 homolog was found in budding yeast, despite the conserved nature of origin Licensing. Here we identify Tah11/Sid2, previously isolated through interactions with topoisomerase and Cdk inhibitor mutants, as an ortholog of Cdt1. We show that sid2 mutants lose minichromosomes in an ARS number-dependent manner, consistent with ScCdt1/Sid2 being involved in origin Licensing. Accordingly, cells partially depleted of Cdt1 replicate DNA from fewer origins, whereas fully depleted cells fail to load Mcm2 on chromatin and fail to initiate but not elongate DNA synthesis. We conclude that origin Licensing depends in S. cerevisiae as in other eukaryotes on both Cdc6 and Cdt1.

P. Todd Stukenberg - One of the best experts on this subject based on the ideXlab platform.