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Anne-sophie Vercoutter-edouart - One of the best experts on this subject based on the ideXlab platform.
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O-GlcNAc transferase associates with the MCM2–7 complex and its silencing destabilizes MCM–MCM interactions
Cellular and Molecular Life Sciences, 2018Co-Authors: Maïté Leturcq, Marlène Mortuaire, Stéphan Hardivillé, Céline Schulz, Tony Lefebvre, Anne-sophie Vercoutter-edouartAbstract:O -GlcNAcylation of proteins is governed by O -GlcNAc transferase (OGT) and O -GlcNAcase (OGA). The homeostasis of O -GlcNAc cycling is regulated during cell cycle progression and is essential for proper cellular division. We previously reported the O -GlcNAcylation of the minichromosome maintenance proteins MCM2, MCM3, MCM6 and MCM7. These proteins belong to the MCM2–7 complex which is crucial for the initiation of DNA replication through its DNA helicase activity. Here we show that the six subunits of MCM2–7 are O -GlcNAcylated and that O -GlcNAcylation of MCM proteins mainly occurs in the chromatin-bound fraction of synchronized human cells. Moreover, we identify stable interaction between OGT and several MCM subunits. We also show that down-regulation of OGT decreases the chromatin binding of MCM2, MCM6 and MCM7 without affecting their steady-state level. Finally, OGT silencing or OGA inhibition destabilizes MCM2/6 and MCM4/7 interactions in the chromatin-enriched fraction. In conclusion, OGT is a new partner of the MCM2–7 complex and O -GlcNAcylation homeostasis might regulate MCM2–7 complex by regulating the chromatin loading of MCM6 and MCM7 and stabilizing MCM/MCM interactions.
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O-GlcNAc transferase associates with the MCM2-7 complex and its silencing destabilizes MCM-MCM interactions
Cellular and Molecular Life Sciences Cmls, 2018Co-Authors: Maïté Leturcq, Marlène Mortuaire, Stéphan Hardivillé, Céline Schulz, Tony Lefebvre, Anne-sophie Vercoutter-edouartAbstract:O-GlcNAcylation of proteins is governed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). The homeostasis of O-GlcNAc cycling is regulated during cell cycle progression and is essential for proper cellular division. We previously reported the O-GlcNAcylation of the Mini-Chromosome Maintenance proteins MCM2, MCM3, MCM6 and MCM7. These proteins belong to the MCM2-7 complex which is crucial for the initiation of DNA replication through its DNA helicase activity. Here we show that the six subunits of MCM2-7 are O-GlcNAcylated and that O-GlcNAcylation of MCM proteins mainly occurs in the chromatin-bound fraction of synchronized human cells. Moreover, we identify stable interaction between OGT and several MCM subunits. We also show that down-regulation of OGT decreases the chromatin binding of MCM2, MCM6 and MCM7 without affecting their steady-state level. Finally, OGT silencing or OGA inhibition destabilize MCM2/6 and MCM4/7 interactions in the chromatin-enriched fraction. In conclusion, OGT is a new partner of the MCM2-7 complex and O-GlcNAcylation homeostasis might regulate MCM2-7 complex by regulating the chromatin loading of MCM6 and MCM7 and stabilizing MCM/MCM interactions.
Maïté Leturcq - One of the best experts on this subject based on the ideXlab platform.
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O-GlcNAc transferase associates with the MCM2–7 complex and its silencing destabilizes MCM–MCM interactions
Cellular and Molecular Life Sciences, 2018Co-Authors: Maïté Leturcq, Marlène Mortuaire, Stéphan Hardivillé, Céline Schulz, Tony Lefebvre, Anne-sophie Vercoutter-edouartAbstract:O -GlcNAcylation of proteins is governed by O -GlcNAc transferase (OGT) and O -GlcNAcase (OGA). The homeostasis of O -GlcNAc cycling is regulated during cell cycle progression and is essential for proper cellular division. We previously reported the O -GlcNAcylation of the minichromosome maintenance proteins MCM2, MCM3, MCM6 and MCM7. These proteins belong to the MCM2–7 complex which is crucial for the initiation of DNA replication through its DNA helicase activity. Here we show that the six subunits of MCM2–7 are O -GlcNAcylated and that O -GlcNAcylation of MCM proteins mainly occurs in the chromatin-bound fraction of synchronized human cells. Moreover, we identify stable interaction between OGT and several MCM subunits. We also show that down-regulation of OGT decreases the chromatin binding of MCM2, MCM6 and MCM7 without affecting their steady-state level. Finally, OGT silencing or OGA inhibition destabilizes MCM2/6 and MCM4/7 interactions in the chromatin-enriched fraction. In conclusion, OGT is a new partner of the MCM2–7 complex and O -GlcNAcylation homeostasis might regulate MCM2–7 complex by regulating the chromatin loading of MCM6 and MCM7 and stabilizing MCM/MCM interactions.
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O-GlcNAc transferase associates with the MCM2-7 complex and its silencing destabilizes MCM-MCM interactions
Cellular and Molecular Life Sciences Cmls, 2018Co-Authors: Maïté Leturcq, Marlène Mortuaire, Stéphan Hardivillé, Céline Schulz, Tony Lefebvre, Anne-sophie Vercoutter-edouartAbstract:O-GlcNAcylation of proteins is governed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). The homeostasis of O-GlcNAc cycling is regulated during cell cycle progression and is essential for proper cellular division. We previously reported the O-GlcNAcylation of the Mini-Chromosome Maintenance proteins MCM2, MCM3, MCM6 and MCM7. These proteins belong to the MCM2-7 complex which is crucial for the initiation of DNA replication through its DNA helicase activity. Here we show that the six subunits of MCM2-7 are O-GlcNAcylated and that O-GlcNAcylation of MCM proteins mainly occurs in the chromatin-bound fraction of synchronized human cells. Moreover, we identify stable interaction between OGT and several MCM subunits. We also show that down-regulation of OGT decreases the chromatin binding of MCM2, MCM6 and MCM7 without affecting their steady-state level. Finally, OGT silencing or OGA inhibition destabilize MCM2/6 and MCM4/7 interactions in the chromatin-enriched fraction. In conclusion, OGT is a new partner of the MCM2-7 complex and O-GlcNAcylation homeostasis might regulate MCM2-7 complex by regulating the chromatin loading of MCM6 and MCM7 and stabilizing MCM/MCM interactions.
Narendra Tuteja - One of the best experts on this subject based on the ideXlab platform.
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Plant MCM proteins: role in DNA replication and beyond
Plant Molecular Biology, 2011Co-Authors: Narendra Tuteja, Hung Quang Dang, Ngoc Quang Tran, Renu TutejaAbstract:Mini-chromosome maintenance (MCM) proteins form heterohexameric complex (MCM2–7) to serve as licensing factor for DNA replication to make sure that genomic DNA is replicated completely and accurately once during S phase in a single cell cycle. MCMs were initially identified in yeast for their role in plasmid replication or cell cycle progression. Each of six MCM contains highly conserved sequence called “MCM box”, which contains two ATPase consensus Walker A and Walker B motifs. Studies on MCM proteins showed that (a) the replication origins are licensed by stable binding of MCM2–7 to form pre-RC (pre-replicative complex) during G1 phase of the cell cycle, (b) the activation of MCM proteins by CDKs (cyclin-dependent kinases) and DDKs (Dbf4-dependent kinases) and their helicase activity are important for pre-RC to initiate the DNA replication, and (c) the release of MCMs from chromatin renders the origins “unlicensed”. DNA replication licensing in plant is, in general, less characterized. The MCMs have been reported from Arabidopsis, maize, tobacco, pea and rice, where they are found to be highly expressed in dividing tissues such as shoot apex and root tips, localized in nucleus and cytosol and play important role in DNA replication, megagametophyte and embryo development. The identification of six MCM coding genes from pea and Arabidopsis suggest six distinct classes of MCM protein in higher plant, and the conserved function right across the eukaryotes. This overview of MCMs contains an emphasis on MCMs from plants and the novel role of MCM6 in abiotic stress tolerance.
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A single subunit MCM6 from pea promotes salinity stress tolerance without affecting yield
Plant Molecular Biology, 2011Co-Authors: Hung Quang Dang, Ngoc Quang Tran, Renu Tuteja, Sarvajeet Singh Gill, Narendra TutejaAbstract:The eukaryotic pre-replicative complex (Pre-RC), including heterohexameric minichromosome maintenance (MCM2-7) proteins, ensures that the DNA in genome is replicated only once per cell division cycle. The MCMs provide DNA unwinding function during the DNA replication. Since MCM proteins play essential role in cell division and most likely are affected during stress conditions therefore their overexpression in plants may help in stress tolerance. But the role of MCMs in abiotic stress tolerance in plants has not been reported so far. In this study we report that: a) the MCM6 transcript is upregulated in pea plant in response to high salinity and cold stress and not with ABA, drought and heat stress; b) MCM6 overexpression driven by a constitutive cauliflower mosaic virus-35S promoter in tobacco plants confers salinity tolerance. The T_1 transgenics plants were able to grow to maturity and set normal viable seeds under continuous salinity stress, without yield penalty. It was observed that in salt-grown T_1 transgenic plants the Na^+ ions is mostly accumulated in mature leaves and not in seeds of T_1 transgenic lines as compared with the wild-type (WT) plants. T_1 transgenic plants exhibited better growth status under salinity stress conditions in comparison to WT plants. Furthermore, the T_1 transgenic plants maintained significantly higher levels of leaf chlorophyll content, net photosynthetic rate and therefore higher dry matter accumulation and yield with 200 mM NaCl as compared to the WT plants. Tolerance index data showed better salt tolerance potential of T_1 transgenic plants in comparison to WT. These findings provide first direct evidence that overexpression of single subunit MCM6 confers salinity stress tolerance without yield loss. The possible mechanism of salinity tolerance is discussed. These findings suggest that DNA replication machinery can be exploited for promoting stress tolerance in crop plants.
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A single subunit MCM6 from pea forms homohexamer and functions as DNA helicase
Plant Molecular Biology, 2010Co-Authors: Ngoc Quang Tran, Hung Quang Dang, Renu Tuteja, Narendra TutejaAbstract:The initiation of DNA replication starts from origins and is controlled by a multiprotein complex, which involves about twenty protein factors. One of the important factors is hetrohexameric minichromosome maintenance (MCM2-7) protein complex which is evolutionary conserved and functions as essential replicative helicase for DNA replication. Here we report the isolation and characterization of a single subunit of pea MCM protein complex, the MCM6. The deduced amino acid (827) sequence contains all the known canonical MCM motifs including zinc finger, MCM specific Walker A and Walker B and arginine finger. The purified recombinant protein contains ATP-dependent 3′–5′ DNA helicase, ATP-binding and ATPase activities. The helicase activity was stimulated by replication fork like substrate and anti-MCM6 antibodies curtail all the enzyme activities of MCM6 protein. In vitro it self-interacts and forms a homohexamer which is active for DNA helicase and ATPase activities. The complete protein is required for self-interaction as the truncated MCM6 proteins were unable to self-interact. Western blot analysis and in vivo immunostaining followed by confocal microscopy showed the localization of MCM6 both in the nucleus and cytosol. These findings provide first direct evidence that single subunit MCM6 contains DNA helicase activity which is unique to plant MCM6 protein, as this activity was only reported for heteromultimers of MCM proteins in animal system. This discovery should make an important contribution to a better understanding of DNA replication in plants.
Hung Quang Dang - One of the best experts on this subject based on the ideXlab platform.
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Plant MCM proteins: role in DNA replication and beyond
Plant Molecular Biology, 2011Co-Authors: Narendra Tuteja, Hung Quang Dang, Ngoc Quang Tran, Renu TutejaAbstract:Mini-chromosome maintenance (MCM) proteins form heterohexameric complex (MCM2–7) to serve as licensing factor for DNA replication to make sure that genomic DNA is replicated completely and accurately once during S phase in a single cell cycle. MCMs were initially identified in yeast for their role in plasmid replication or cell cycle progression. Each of six MCM contains highly conserved sequence called “MCM box”, which contains two ATPase consensus Walker A and Walker B motifs. Studies on MCM proteins showed that (a) the replication origins are licensed by stable binding of MCM2–7 to form pre-RC (pre-replicative complex) during G1 phase of the cell cycle, (b) the activation of MCM proteins by CDKs (cyclin-dependent kinases) and DDKs (Dbf4-dependent kinases) and their helicase activity are important for pre-RC to initiate the DNA replication, and (c) the release of MCMs from chromatin renders the origins “unlicensed”. DNA replication licensing in plant is, in general, less characterized. The MCMs have been reported from Arabidopsis, maize, tobacco, pea and rice, where they are found to be highly expressed in dividing tissues such as shoot apex and root tips, localized in nucleus and cytosol and play important role in DNA replication, megagametophyte and embryo development. The identification of six MCM coding genes from pea and Arabidopsis suggest six distinct classes of MCM protein in higher plant, and the conserved function right across the eukaryotes. This overview of MCMs contains an emphasis on MCMs from plants and the novel role of MCM6 in abiotic stress tolerance.
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the cdc45 mcm2 7 gins protein complex in trypanosomes regulates dna replication and interacts with two orc1 like proteins in the origin recognition complex
Journal of Biological Chemistry, 2011Co-Authors: Hung Quang Dang, Ziyin LiAbstract:Accurate DNA replication requires a complex interplay of many regulatory proteins at replication origins. The CMG (Cdc45·Mcm2–7·GINS) complex, which is composed of Cdc45, Mcm2–7, and the GINS (Go-Ichi-Ni-San) complex consisting of Sld5 and Psf1 to Psf3, is recruited by Cdc6 and Cdt1 onto origins bound by the heterohexameric origin recognition complex (ORC) and functions as a replicative helicase. Trypanosoma brucei, an early branched microbial eukaryote, appears to express an archaea-like ORC consisting of a single Orc1/Cdc6-like protein. However, unlike archaea, trypanosomes possess components of the eukaryote-like CMG complex, but whether they form an active helicase complex, associate with the ORC, and regulate DNA replication remains unknown. Here, we demonstrated that the CMG complex is formed in vivo in trypanosomes and that Mcm2–7 helicase activity is activated by the association with Cdc45 and the GINS complex in vitro. Mcm2–7 and GINS proteins are confined to the nucleus throughout the cell cycle, whereas Cdc45 is exported out of the nucleus after DNA replication, indicating that nuclear exclusion of Cdc45 constitutes one mechanism for preventing DNA re-replication in trypanosomes. With the exception of Mcm4, Mcm6, and Psf1, knockdown of individual CMG genes inhibits DNA replication and cell proliferation. Finally, we identified a novel Orc1-like protein, Orc1b, as an additional component of the ORC and showed that both Orc1b and Orc1/Cdc6 associate with Mcm2–7 via interactions with Mcm3. All together, we identified the Cdc45·Mcm2–7·GINS complex as the replicative helicase that interacts with two Orc1-like proteins in the unusual origin recognition complex in trypanosomes.
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A single subunit MCM6 from pea promotes salinity stress tolerance without affecting yield
Plant Molecular Biology, 2011Co-Authors: Hung Quang Dang, Ngoc Quang Tran, Renu Tuteja, Sarvajeet Singh Gill, Narendra TutejaAbstract:The eukaryotic pre-replicative complex (Pre-RC), including heterohexameric minichromosome maintenance (MCM2-7) proteins, ensures that the DNA in genome is replicated only once per cell division cycle. The MCMs provide DNA unwinding function during the DNA replication. Since MCM proteins play essential role in cell division and most likely are affected during stress conditions therefore their overexpression in plants may help in stress tolerance. But the role of MCMs in abiotic stress tolerance in plants has not been reported so far. In this study we report that: a) the MCM6 transcript is upregulated in pea plant in response to high salinity and cold stress and not with ABA, drought and heat stress; b) MCM6 overexpression driven by a constitutive cauliflower mosaic virus-35S promoter in tobacco plants confers salinity tolerance. The T_1 transgenics plants were able to grow to maturity and set normal viable seeds under continuous salinity stress, without yield penalty. It was observed that in salt-grown T_1 transgenic plants the Na^+ ions is mostly accumulated in mature leaves and not in seeds of T_1 transgenic lines as compared with the wild-type (WT) plants. T_1 transgenic plants exhibited better growth status under salinity stress conditions in comparison to WT plants. Furthermore, the T_1 transgenic plants maintained significantly higher levels of leaf chlorophyll content, net photosynthetic rate and therefore higher dry matter accumulation and yield with 200 mM NaCl as compared to the WT plants. Tolerance index data showed better salt tolerance potential of T_1 transgenic plants in comparison to WT. These findings provide first direct evidence that overexpression of single subunit MCM6 confers salinity stress tolerance without yield loss. The possible mechanism of salinity tolerance is discussed. These findings suggest that DNA replication machinery can be exploited for promoting stress tolerance in crop plants.
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A single subunit MCM6 from pea forms homohexamer and functions as DNA helicase
Plant Molecular Biology, 2010Co-Authors: Ngoc Quang Tran, Hung Quang Dang, Renu Tuteja, Narendra TutejaAbstract:The initiation of DNA replication starts from origins and is controlled by a multiprotein complex, which involves about twenty protein factors. One of the important factors is hetrohexameric minichromosome maintenance (MCM2-7) protein complex which is evolutionary conserved and functions as essential replicative helicase for DNA replication. Here we report the isolation and characterization of a single subunit of pea MCM protein complex, the MCM6. The deduced amino acid (827) sequence contains all the known canonical MCM motifs including zinc finger, MCM specific Walker A and Walker B and arginine finger. The purified recombinant protein contains ATP-dependent 3′–5′ DNA helicase, ATP-binding and ATPase activities. The helicase activity was stimulated by replication fork like substrate and anti-MCM6 antibodies curtail all the enzyme activities of MCM6 protein. In vitro it self-interacts and forms a homohexamer which is active for DNA helicase and ATPase activities. The complete protein is required for self-interaction as the truncated MCM6 proteins were unable to self-interact. Western blot analysis and in vivo immunostaining followed by confocal microscopy showed the localization of MCM6 both in the nucleus and cytosol. These findings provide first direct evidence that single subunit MCM6 contains DNA helicase activity which is unique to plant MCM6 protein, as this activity was only reported for heteromultimers of MCM proteins in animal system. This discovery should make an important contribution to a better understanding of DNA replication in plants.
Marlène Mortuaire - One of the best experts on this subject based on the ideXlab platform.
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O-GlcNAc transferase associates with the MCM2–7 complex and its silencing destabilizes MCM–MCM interactions
Cellular and Molecular Life Sciences, 2018Co-Authors: Maïté Leturcq, Marlène Mortuaire, Stéphan Hardivillé, Céline Schulz, Tony Lefebvre, Anne-sophie Vercoutter-edouartAbstract:O -GlcNAcylation of proteins is governed by O -GlcNAc transferase (OGT) and O -GlcNAcase (OGA). The homeostasis of O -GlcNAc cycling is regulated during cell cycle progression and is essential for proper cellular division. We previously reported the O -GlcNAcylation of the minichromosome maintenance proteins MCM2, MCM3, MCM6 and MCM7. These proteins belong to the MCM2–7 complex which is crucial for the initiation of DNA replication through its DNA helicase activity. Here we show that the six subunits of MCM2–7 are O -GlcNAcylated and that O -GlcNAcylation of MCM proteins mainly occurs in the chromatin-bound fraction of synchronized human cells. Moreover, we identify stable interaction between OGT and several MCM subunits. We also show that down-regulation of OGT decreases the chromatin binding of MCM2, MCM6 and MCM7 without affecting their steady-state level. Finally, OGT silencing or OGA inhibition destabilizes MCM2/6 and MCM4/7 interactions in the chromatin-enriched fraction. In conclusion, OGT is a new partner of the MCM2–7 complex and O -GlcNAcylation homeostasis might regulate MCM2–7 complex by regulating the chromatin loading of MCM6 and MCM7 and stabilizing MCM/MCM interactions.
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O-GlcNAc transferase associates with the MCM2-7 complex and its silencing destabilizes MCM-MCM interactions
Cellular and Molecular Life Sciences Cmls, 2018Co-Authors: Maïté Leturcq, Marlène Mortuaire, Stéphan Hardivillé, Céline Schulz, Tony Lefebvre, Anne-sophie Vercoutter-edouartAbstract:O-GlcNAcylation of proteins is governed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). The homeostasis of O-GlcNAc cycling is regulated during cell cycle progression and is essential for proper cellular division. We previously reported the O-GlcNAcylation of the Mini-Chromosome Maintenance proteins MCM2, MCM3, MCM6 and MCM7. These proteins belong to the MCM2-7 complex which is crucial for the initiation of DNA replication through its DNA helicase activity. Here we show that the six subunits of MCM2-7 are O-GlcNAcylated and that O-GlcNAcylation of MCM proteins mainly occurs in the chromatin-bound fraction of synchronized human cells. Moreover, we identify stable interaction between OGT and several MCM subunits. We also show that down-regulation of OGT decreases the chromatin binding of MCM2, MCM6 and MCM7 without affecting their steady-state level. Finally, OGT silencing or OGA inhibition destabilize MCM2/6 and MCM4/7 interactions in the chromatin-enriched fraction. In conclusion, OGT is a new partner of the MCM2-7 complex and O-GlcNAcylation homeostasis might regulate MCM2-7 complex by regulating the chromatin loading of MCM6 and MCM7 and stabilizing MCM/MCM interactions.