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Richard Mcculloch - One of the best experts on this subject based on the ideXlab platform.

  • identification of orc1 cdc6 interacting factors in trypanosoma brucei reveals critical features of origin recognition Complex architecture
    PLOS ONE, 2012
    Co-Authors: Calvin Tiengwe, Lucio Marcello, Helen Farr, Catarina Gadelha, Richard Burchmore, David J Barry, Stephen D Bell, Richard Mcculloch
    Abstract:

    DNA Replication initiates by formation of a Pre-Replication Complex on sequences termed origins. In eukaryotes, the Pre-Replication Complex is composed of the Origin Recognition Complex (ORC), Cdc6 and the MCM replicative helicase in conjunction with Cdt1. Eukaryotic ORC is considered to be composed of six subunits, named Orc1–6, and monomeric Cdc6 is closely related in sequence to Orc1. However, ORC has been little explored in protists, and only a single ORC protein, related to both Orc1 and Cdc6, has been shown to act in DNA replication in Trypanosoma brucei. Here we identify three highly diverged putative T. brucei ORC components that interact with ORC1/CDC6 and contribute to cell division. Two of these factors are so diverged that we cannot determine if they are eukaryotic ORC subunit orthologues, or are parasite-specific replication factors. The other we show to be a highly diverged Orc4 orthologue, demonstrating that this is one of the most widely conserved ORC subunits in protists and revealing it to be a key element of eukaryotic ORC architecture. Additionally, we have examined interactions amongst the T. brucei MCM subunits and show that this has the conventional eukaryotic heterohexameric structure, suggesting that divergence in the T. brucei replication machinery is limited to the earliest steps in origin licensing.

  • Identification of ORC1/CDC6-Interacting Factors in Trypanosoma brucei Reveals Critical Features of Origin Recognition Complex Architecture
    PloS one, 2012
    Co-Authors: Calvin Tiengwe, Lucio Marcello, Helen Farr, Catarina Gadelha, Richard Burchmore, Stephen D Bell, J. David Barry, Richard Mcculloch
    Abstract:

    DNA Replication initiates by formation of a Pre-Replication Complex on sequences termed origins. In eukaryotes, the Pre-Replication Complex is composed of the Origin Recognition Complex (ORC), Cdc6 and the MCM replicative helicase in conjunction with Cdt1. Eukaryotic ORC is considered to be composed of six subunits, named Orc1–6, and monomeric Cdc6 is closely related in sequence to Orc1. However, ORC has been little explored in protists, and only a single ORC protein, related to both Orc1 and Cdc6, has been shown to act in DNA replication in Trypanosoma brucei. Here we identify three highly diverged putative T. brucei ORC components that interact with ORC1/CDC6 and contribute to cell division. Two of these factors are so diverged that we cannot determine if they are eukaryotic ORC subunit orthologues, or are parasite-specific replication factors. The other we show to be a highly diverged Orc4 orthologue, demonstrating that this is one of the most widely conserved ORC subunits in protists and revealing it to be a key element of eukaryotic ORC architecture. Additionally, we have examined interactions amongst the T. brucei MCM subunits and show that this has the conventional eukaryotic heterohexameric structure, suggesting that divergence in the T. brucei replication machinery is limited to the earliest steps in origin licensing.

Melvin L. Depamphilis - One of the best experts on this subject based on the ideXlab platform.

  • Site-specific ORC binding, Pre-Replication Complex assembly and DNA synthesis at Schizosaccharomyces pombe replication origins
    The EMBO journal, 2002
    Co-Authors: Daochun Kong, Melvin L. Depamphilis
    Abstract:

    Previous studies have shown that the Schizo saccharomyces pombe Orc4 subunit is solely responsible for in vitro binding of origin recognition Complex (ORC) to specific AT-rich sites within S.pombe replication origins. Using ARS3001, a S.pombe replication origin consisting of four genetically required sites, we show that, in situ as well as in vitro, Orc4 binds strongly to the Δ3 site, weakly to the Δ6 site and not at all to the remaining sequences. In situ, the footprint over Δ3 is extended during G1 phase, but only when Cdc18 is present and Mcm proteins are bound to chromatin. Moreover, this footprint extends into the adjacent Δ2 site, where leading strand DNA synthesis begins. Therefore, we conclude that ARS3001 consists of a single primary ORC binding site that assembles a Pre-Replication Complex and initiates DNA synthesis, plus an additional novel origin element (Δ9) that neither binds ORC nor functions as a centromere, but does bind an as yet unidentified protein throughout the cell cycle. Schizosaccharomyces pombe may be an appropriate paradigm for the Complex origins found in the metazoa.

  • Selective instability of Orc1 protein accounts for the absence of functional origin recognition Complexes during the M–G1 transition in mammals
    The EMBO journal, 2000
    Co-Authors: Darren A. Natale, Wei-hsin Sun, Melvin L. Depamphilis
    Abstract:

    To investigate the events leading to initiation of DNA replication in mammalian chromosomes, the time when hamster origin recognition Complexes (ORCs) became functional was related to the time when Orc1, Orc2 and Mcm3 proteins became stably bound to hamster chromatin. Functional ORCs, defined as those able to initiate DNA replication, were absent during mitosis and early G1 phase, and reappeared as cells progressed through G1 phase. Immunoblotting analysis revealed that hamster Orc1 and Orc2 proteins were present in nuclei at equivalent concentrations throughout the cell cycle, but only Orc2 was stably bound to chromatin. Orc1 and Mcm3 were easily eluted from chromatin during mitosis and early G1 phase, but became stably bound during mid-G1 phase, concomitant with the appearance of a functional Pre-Replication Complex at a hamster replication origin. Since hamster Orc proteins are closely related to their human and mouse homologs, the unexpected behavior of hamster Orc1 provides a novel mechanism in mammals for delaying assembly of Pre-Replication Complexes until mitosis is complete and a nuclear structure has formed.

  • Replication origins in metazoan chromosomes: fact or fiction?
    BioEssays, 1999
    Co-Authors: Melvin L. Depamphilis
    Abstract:

    The process by which eukaryotic cells decide when and where to initiate DNA replication has been illuminated in yeast, where specific DNA sequences (replication origins) bind a unique group of proteins (origin recognition Complex) next to an easily unwound DNA sequence at which replication can begin. The origin recognition Complex provides a platform on which additional proteins assemble to form a Pre-Replication Complex that can be activated at S-phase by specific protein kinases. Remarkably, multicellular eukaryotes, such as frogs, flies, and mammals (metazoa), have counterparts to these yeast proteins that are required for DNA replication. Therefore, one might expect metazoan chromosomes to contain specific replication origins as well, a hypothesis that has long been controversial. In fact, recent results strongly support the view that DNA replication origins in metazoan chromosomes consist of one or more high frequency initiation sites and perhaps several low frequency ones that together can appear as a nonspecific initiation zone. Specific replication origins are established during G1-phase of each cell cycle by multiple parameters that include nuclear structure, chromatin structure, DNA sequence, and perhaps DNA modification. Such Complexity endows metazoa with the flexibility to change both the number and locations of replication origins in response to the demands of animal development. BioEssays 1999;21:5–16. © 1999 John Wiley & Sons, Inc.

Calvin Tiengwe - One of the best experts on this subject based on the ideXlab platform.

  • identification of orc1 cdc6 interacting factors in trypanosoma brucei reveals critical features of origin recognition Complex architecture
    PLOS ONE, 2012
    Co-Authors: Calvin Tiengwe, Lucio Marcello, Helen Farr, Catarina Gadelha, Richard Burchmore, David J Barry, Stephen D Bell, Richard Mcculloch
    Abstract:

    DNA Replication initiates by formation of a Pre-Replication Complex on sequences termed origins. In eukaryotes, the Pre-Replication Complex is composed of the Origin Recognition Complex (ORC), Cdc6 and the MCM replicative helicase in conjunction with Cdt1. Eukaryotic ORC is considered to be composed of six subunits, named Orc1–6, and monomeric Cdc6 is closely related in sequence to Orc1. However, ORC has been little explored in protists, and only a single ORC protein, related to both Orc1 and Cdc6, has been shown to act in DNA replication in Trypanosoma brucei. Here we identify three highly diverged putative T. brucei ORC components that interact with ORC1/CDC6 and contribute to cell division. Two of these factors are so diverged that we cannot determine if they are eukaryotic ORC subunit orthologues, or are parasite-specific replication factors. The other we show to be a highly diverged Orc4 orthologue, demonstrating that this is one of the most widely conserved ORC subunits in protists and revealing it to be a key element of eukaryotic ORC architecture. Additionally, we have examined interactions amongst the T. brucei MCM subunits and show that this has the conventional eukaryotic heterohexameric structure, suggesting that divergence in the T. brucei replication machinery is limited to the earliest steps in origin licensing.

  • Identification of ORC1/CDC6-Interacting Factors in Trypanosoma brucei Reveals Critical Features of Origin Recognition Complex Architecture
    PloS one, 2012
    Co-Authors: Calvin Tiengwe, Lucio Marcello, Helen Farr, Catarina Gadelha, Richard Burchmore, Stephen D Bell, J. David Barry, Richard Mcculloch
    Abstract:

    DNA Replication initiates by formation of a Pre-Replication Complex on sequences termed origins. In eukaryotes, the Pre-Replication Complex is composed of the Origin Recognition Complex (ORC), Cdc6 and the MCM replicative helicase in conjunction with Cdt1. Eukaryotic ORC is considered to be composed of six subunits, named Orc1–6, and monomeric Cdc6 is closely related in sequence to Orc1. However, ORC has been little explored in protists, and only a single ORC protein, related to both Orc1 and Cdc6, has been shown to act in DNA replication in Trypanosoma brucei. Here we identify three highly diverged putative T. brucei ORC components that interact with ORC1/CDC6 and contribute to cell division. Two of these factors are so diverged that we cannot determine if they are eukaryotic ORC subunit orthologues, or are parasite-specific replication factors. The other we show to be a highly diverged Orc4 orthologue, demonstrating that this is one of the most widely conserved ORC subunits in protists and revealing it to be a key element of eukaryotic ORC architecture. Additionally, we have examined interactions amongst the T. brucei MCM subunits and show that this has the conventional eukaryotic heterohexameric structure, suggesting that divergence in the T. brucei replication machinery is limited to the earliest steps in origin licensing.

Bruce Stillman - One of the best experts on this subject based on the ideXlab platform.

  • a double hexameric mcm2 7 Complex is loaded onto origin dna during licensing of eukaryotic dna replication
    Proceedings of the National Academy of Sciences of the United States of America, 2009
    Co-Authors: Cecile Evrin, Bruce Stillman, Pippa Clarke, Juergen Zech, Rudi Lurz, Stefan Uhle, H Li, Christian Speck
    Abstract:

    During Pre-Replication Complex (pre-RC) formation, origin recognition Complex (ORC), Cdc6, and Cdt1 cooperatively load the 6-subunit mini chromosome maintenance (MCM2-7) Complex onto DNA. Loading of MCM2-7 is a prerequisite for DNA licensing that restricts DNA replication to once per cell cycle. During S phase MCM2-7 functions as part of the replicative helicase but within the pre-RC MCM2-7 is inactive. The organization of replicative DNA helicases before and after loading onto DNA has been studied in bacteria and viruses but not eukaryotes and is of major importance for understanding the MCM2-7 loading mechanism and replisome assembly. Lack of an efficient reconstituted pre-RC system has hindered the detailed mechanistic and structural analysis of MCM2-7 loading for a long time. We have reconstituted Saccharomyces cerevisiae pre-RC formation with purified proteins and showed efficient loading of MCM2-7 onto origin DNA in vitro. MCM2-7 loading was found to be dependent on the presence of all pre-RC proteins, origin DNA, and ATP hydrolysis. The quaternary structure of MCM2-7 changes during pre-RC formation: MCM2-7 before loading is a single hexamer in solution but is transformed into a double-hexamer during pre-RC formation. Using electron microscopy (EM), we observed that loaded MCM2-7 encircles DNA. The loaded MCM2-7 Complex can slide on DNA, and sliding is not directional. Our results provide key insights into mechanisms of pre-RC formation and have important implications for understanding the role of the MCM2-7 in establishment of bidirectional replication forks.

  • ATPase-dependent cooperative binding of ORC and Cdc6 to origin DNA
    Nature Structural & Molecular Biology, 2005
    Co-Authors: Christian Speck, Huilin Li, Zhiqiang Chen, Bruce Stillman
    Abstract:

    Binding of Cdc6 to the origin recognition Complex (ORC) is a key step in the assembly of a Pre-Replication Complex (pre-RC) at origins of DNA replication. ORC recognizes specific origin DNA sequences in an ATP-dependent manner. Here we demonstrate cooperative binding of Saccharomyces cerevisiae Cdc6 to ORC on DNA in an ATP-dependent manner, which induces a change in the pattern of origin binding that requires the Orc1 ATPase. The reaction is blocked by specific origin mutations that do not interfere with the interaction between ORC and DNA. Single-particle reconstruction of electron microscopic images shows that the ORC–Cdc6 Complex forms a ring-shaped structure with dimensions similar to those of the ring-shaped MCM helicase. The ORC-Cdc6 structure is predicted to contain six AAA+ subunits, analogous to other ATP-dependent protein machines. We suggest that Cdc6 and origin DNA activate a molecular switch in ORC that contributes to pre-RC assembly.

  • Dynamics of Pre-Replication Complex proteins during the cell division cycle.
    Philosophical transactions of the Royal Society of London. Series B Biological sciences, 2004
    Co-Authors: Supriya G. Prasanth, Juan Méndez, Kannanganattu V. Prasanth, Bruce Stillman
    Abstract:

    Replication of the human genome every time a cell divides is a highly coordinated process that ensures accurate and efficient inheritance of the genetic information. The molecular mechanism that guarantees that many origins of replication fire only once per cell-cycle has been the area of intense research. The origin recognition Complex (ORC) marks the position of replication origins in the genome and serves as the landing pad for the assembly of a multiprotein, pre-replicative Complex (pre-RC) at the origins, consisting of ORC, cell division cycle 6 (Cdc6), Cdc10-dependent transcript (Cdt1) and mini-chromosome maintenance (MCM) proteins. The MCM proteins serve as key participants in the mechanism that limits eukaryotic DNA replication to once-per-cell-cycle and its binding to the chromatin marks the final step of pre-RC formation, a process referred to as 'replication licensing'. We present data demonstrating how the MCM proteins associate with the chromatin during the G1 phase, probably defining pre-RCs and then anticipate replication fork movement in a precisely coordinated manner during the S phase of the cell cycle. The process of DNA replication must also be carefully coordinated with other cell-cycle processes including mitosis and cytokinesis. Some of the proteins that control initiation of DNA replication are likely to interact with the pathways that control these important cell-cycle transitions. Herein, we discuss the participation of human ORC proteins in other vital functions, in addition to their bona fide roles in replication.

Masatoshi Fujita - One of the best experts on this subject based on the ideXlab platform.

  • Nucleosome assembly and disassembly activity of GRWD1, a novel Cdt1-binding protein that promotes Pre-Replication Complex formation.
    Biochimica et biophysica acta, 2016
    Co-Authors: Masahiro Aizawa, Kazumasa Yoshida, Nozomi Sugimoto, Shinya Watanabe, Masatoshi Fujita
    Abstract:

    GRWD1 was previously identified as a novel Cdt1-binding protein that possesses histone-binding and nucleosome assembly activities and promotes MCM loading, probably by maintaining chromatin openness at replication origins. However, the molecular mechanisms underlying these activities remain unknown. We prepared reconstituted mononucleosomes from recombinant histones and a DNA fragment containing a nucleosome positioning sequence, and investigated the effects of GRWD1 on them. GRWD1 could disassemble these preformed mononucleosomes in vitro in an ATP-independent manner. Thus, our data suggest that GRWD1 facilitates removal of H2A-H2B dimers from nucleosomes, resulting in formation of hexasomes. The activity was compromised by deletion of the acidic domain, which is required for efficient histone binding. In contrast, nucleosome assembly activity of GRWD1 was not affected by deletion of the acidic domain. In HeLa cells, the acidic domain of GRWD1 was necessary to maintain chromatin openness and promote MCM loading at replication origins. Taken together, our results suggest that GRWD1 promotes chromatin fluidity by influencing nucleosome structures, e.g., by transient eviction of H2A-H2B, and thereby promotes efficient MCM loading at replication origins.

  • Chromatin Remodeler Sucrose Nonfermenting 2 Homolog (SNF2H) Is Recruited onto DNA Replication Origins through Interaction with Cdc10 Protein-dependent Transcript 1 (Cdt1) and Promotes Pre-Replication Complex Formation
    Journal of Biological Chemistry, 2011
    Co-Authors: Nozomi Sugimoto, Takashi Yugawa, Tohru Kiyono, Masayoshi Iizuka, Masatoshi Fujita
    Abstract:

    Abstract From late mitosis to the G1 phase of the cell cycle, ORC, CDC6, and Cdt1 form the machinery necessary to load MCM2–7 Complexes onto DNA. Here, we show that SNF2H, a member of the ATP-dependent chromatin-remodeling Complex, is recruited onto DNA replication origins in human cells in a Cdt1-dependent manner and positively regulates MCM loading. SNF2H physically interacted with Cdt1. ChIP assays indicated that SNF2H associates with replication origins specifically during the G1 phase. Binding of SNF2H at origins was decreased by Cdt1 silencing and, conversely, enhanced by Cdt1 overexpression. Furthermore, SNF2H silencing prevented MCM loading at origins and moderately inhibited S phase progression. Although neither SNF2H overexpression nor SNF2H silencing appeared to impact rereplication induced by Cdt1 overexpression, Cdt1-induced checkpoint activation was inhibited by SNF2H silencing. Collectively, these data suggest that SNF2H may promote MCM loading at DNA replication origins via interaction with Cdt1 in human cells. Because efficient loading of excess MCM Complexes is thought to be required for cells to tolerate replication stress, Cdt1- and SNF2H-mediated promotion of MCM loading may be biologically relevant for the regulation of DNA replication.

  • Involvement of human ORC and TRF2 in Pre-Replication Complex assembly at telomeres.
    Genes to cells : devoted to molecular & cellular mechanisms, 2008
    Co-Authors: Yasutoshi Tatsumi, Kai Ezura, Kazumasa Yoshida, Takashi Yugawa, Mako Narisawa-saito, Tohru Kiyono, Satoshi Ohta, Chikashi Obuse, Masatoshi Fujita
    Abstract:

    The origin recognition Complex (ORC) binds to replication origins to regulate the cell cycle-dependent assembly of Pre-Replication Complexes (pre-RCs). We have found a novel link between pre-RC assembly regulation and telomere homeostasis in human cells. Biochemical analyses showed that human ORC binds to TRF2, a telomere sequence-binding protein that protects telomeres and functions in telomere length homeostasis, via the ORC1 subunit. Immunostaining further revealed that ORC and TRF2 partially co-localize in nuclei, whereas chromatin immunoprecipitation analyses confirmed that pre-RCs are assembled at telomeres in a cell cycle-dependent manner. Over-expression of TRF2 stimulated ORC and MCM binding to chromatin and RNAi-directed TRF2 silencing resulted in reduced ORC binding and pre-RC assembly at telomeres. As expected from previous reports, TRF2 silencing induced telomere elongation. Interestingly, ORC1 silencing by RNAi weakened the TRF2 binding as well as the pre-RC assembly at telomeres, suggesting that ORC and TRF2 interact with each other to achieve stable binding. Furthermore, ORC1 silencing also resulted in modest telomere elongation. These data suggest that ORC might be involved in telomere homeostasis in human cells.