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

  • Eukaryotic DNA Replication with purified budding yeast proteins
    Methods in Enzymology, 2021
    Co-Authors: Erik Johansson, Viktor Posse, John F. X. Diffley
    Abstract:

    Abstract The in vitro reconstitution of origin firing was a key step toward the biochemical reconstitution of Eukaryotic DNA Replication in budding yeast. Today the basic Replication assay involves proteins purified from 24 separate protocols that have evolved since their first publication, and as a result, the efficiency and reliability of the in vitro Replication system has improved. Here we will present protocols for all 24 purifications together with a general protocol for the in vitro Replication assay and some tips for troubleshooting problems with the assay.

  • bidirectional Eukaryotic DNA Replication is established by quasi symmetrical helicase loading
    Science, 2017
    Co-Authors: Gideon Coster, John F. X. Diffley
    Abstract:

    Bidirectional Replication from Eukaryotic DNA Replication origins requires the loading of two ring-shaped minichromosome maintenance (MCM) helicases around DNA in opposite orientations. MCM loading is orchestrated by binding of the origin recognition complex (ORC) to DNA, but how ORC coordinates symmetrical MCM loading is unclear. We used natural budding yeast DNA Replication origins and synthetic DNA sequences to show that efficient MCM loading requires binding of two ORC molecules to two ORC binding sites. The relative orientation of these sites, but not the distance between them, was found to be critical for MCM loading in vitro and origin function in vivo. We propose that quasi-symmetrical loading of individual MCM hexamers by ORC and directed MCM translocation into double hexamers acts as a unifying mechanism for the establishment of bidirectional Replication in archaea and eukaryotes.

  • regulated Eukaryotic DNA Replication origin firing with purified proteins
    Nature, 2015
    Co-Authors: Joseph T P Yeeles, Anne Early, Tom D Deegan, Agnieszka Janska, John F. X. Diffley
    Abstract:

    Eukaryotic cells initiate DNA Replication from multiple origins, which must be tightly regulated to promote precise genome duplication in every cell cycle. To accomplish this, initiation is partitioned into two temporally discrete steps: a double hexameric minichromosome maintenance (MCM) complex is first loaded at Replication origins during G1 phase, and then converted to the active CMG (Cdc45–MCM–GINS) helicase during S phase. Here we describe the reconstitution of budding yeast DNA Replication initiation with 16 purified Replication factors, made from 42 polypeptides. Origin-dependent initiation recapitulates regulation seen in vivo. Cyclin-dependent kinase (CDK) inhibits MCM loading by phosphorylating the origin recognition complex (ORC) and promotes CMG formation by phosphorylating Sld2 and Sld3. Dbf4-dependent kinase (DDK) promotes Replication by phosphorylating MCM, and can act either before or after CDK. These experiments define the minimum complement of proteins, protein kinase substrates and co-factors required for regulated Eukaryotic DNA Replication. It has long been a goal to reconstitute Eukaryotic DNA Replication; here a purified in vitro system from budding yeast containing 16 factors, themselves composed of 42 polypeptides, fulfils the staged process of origin-dependent initiation, including its regulation by kinases. It has been a long-desired goal to be able to reconstitute a Eukaryotic system of DNA Replication from its earliest stages of origin firing using purified proteins. However, the greater complexity of eukaryotes compared to bacterial and phage systems has hampered this development. But now John Diffley and colleagues have successfully reconstituted the initial events of budding yeast DNA Replication in vitro. The purified system contains 42 proteins, comprising 16 complexes, and fulfills the staged process of origin-dependent initiation, including its regulation by kinases.

  • regulated Eukaryotic DNA Replication origin firing with purified proteins
    Nature, 2015
    Co-Authors: Joseph T P Yeeles, Anne Early, Tom D Deegan, Agnieszka Janska, John F. X. Diffley
    Abstract:

    Eukaryotic cells initiate DNA Replication from multiple origins, which must be tightly regulated to promote precise genome duplication in every cell cycle. To accomplish this, initiation is partitioned into two temporally discrete steps: a double hexameric minichromosome maintenance (MCM) complex is first loaded at Replication origins during G1 phase, and then converted to the active CMG (Cdc45-MCM-GINS) helicase during S phase. Here we describe the reconstitution of budding yeast DNA Replication initiation with 16 purified Replication factors, made from 42 polypeptides. Origin-dependent initiation recapitulates regulation seen in vivo. Cyclin-dependent kinase (CDK) inhibits MCM loading by phosphorylating the origin recognition complex (ORC) and promotes CMG formation by phosphorylating Sld2 and Sld3. Dbf4-dependent kinase (DDK) promotes Replication by phosphorylating MCM, and can act either before or after CDK. These experiments define the minimum complement of proteins, protein kinase substrates and co-factors required for regulated Eukaryotic DNA Replication.

  • prereplicative complexes assembled in vitro support origin dependent and independent DNA Replication
    The EMBO Journal, 2014
    Co-Authors: Kin Fan On, Fabienne Beuron, Edward P Morris, David Frith, Ambrosius P Snijders, John F. X. Diffley
    Abstract:

    Eukaryotic DNA Replication initiates from multiple Replication origins. To ensure each origin fires just once per cell cycle, initiation is divided into two biochemically discrete steps: the Mcm2-7 helicase is first loaded into prereplicative complexes (pre-RCs) as an inactive double hexamer by the origin recognition complex (ORC), Cdt1 and Cdc6; the helicase is then activated by a set of “firing factors.” Here, we show that plasmids containing pre-RCs assembled with purified proteins support complete and semi-conservative Replication in extracts from budding yeast cells overexpressing firing factors. Replication requires cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK). DDK phosphorylation of Mcm2-7 does not by itself promote separation of the double hexamer, but is required for the recruitment of firing factors and replisome components in the extract. Plasmid Replication does not require a functional Replication origin; however, in the presence of competitor DNA and limiting ORC concentrations, Replication becomes origin-dependent in this system. These experiments indicate that Mcm2-7 double hexamers can be precursors of Replication and provide insight into the nature of Eukaryotic DNA Replication origins.

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

  • The structure of ORC–Cdc6 on an origin DNA reveals the mechanism of ORC activation by the Replication initiator Cdc6
    'Springer Science and Business Media LLC', 2021
    Co-Authors: Xiang Feng, Bruce Stillman, Yasunori Noguchi, Marta Barbon, Christian Speck
    Abstract:

    Eukaryotic DNA Replication is mediated by many proteins which are tightly regulated for an efficient firing of Replication at each cell cycle. Here the authors report a cryo-EM structure of the yeast ORC–Cdc6 bound to an 85-bp ARS1 origin DNA revealing additional insights into how Cdc6 contributes to origin DNA recognition

  • 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.

  • perpetuating the double helix molecular machines at Eukaryotic DNA Replication origins
    BioEssays, 2003
    Co-Authors: Juan Mendez, Bruce Stillman
    Abstract:

    Summary Thehardestpartofreplicatingagenomeisthebeginning. The first step of DNA Replication (called ‘‘initiation’’) mobilizes a large number of specialized proteins (‘‘initiators’’) that recognize specific sequences or structural motifs in the DNA, unwind the double helix, protect the exposed ssDNA, and recruit the enzymatic activities required for DNA synthesis, such as helicases, primases and polymerases. All of these components are orderly assembled before the first nucleotide can be incorporated. On the occasion of the 50th anniversary of the discovery of the DNA structure, we review our current knowledge of the molecular mechanisms that control initiation of DNA Replication in Eukaryotic cells, with particular emphasis on the recent identification of novel initiator proteins. We speculate how these initiators assemble molecular machines capable of performing specific biochemical tasks, such as loading a ringshaped helicase onto the DNA double helix. BioEssays 25:1158–1167, 2003. 2003 Wiley Periodicals, Inc.

  • perpetuating the double helix molecular machines at Eukaryotic DNA Replication origins
    BioEssays, 2003
    Co-Authors: Juan Mendez, Bruce Stillman
    Abstract:

    The hardest part of replicating a genome is the beginning. The first step of DNA Replication (called "initiation") mobilizes a large number of specialized proteins ("initiators") that recognize specific sequences or structural motifs in the DNA, unwind the double helix, protect the exposed ssDNA, and recruit the enzymatic activities required for DNA synthesis, such as helicases, primases and polymerases. All of these components are orderly assembled before the first nucleotide can be incorporated. On the occasion of the 50th anniversary of the discovery of the DNA structure, we review our current knowledge of the molecular mechanisms that control initiation of DNA Replication in Eukaryotic cells, with particular emphasis on the recent identification of novel initiator proteins. We speculate how these initiators assemble molecular machines capable of performing specific biochemical tasks, such as loading a ring-shaped helicase onto the DNA double helix.

  • identification of Eukaryotic DNA Replication proteins using simian virus 40 in vitro Replication system
    Methods in Enzymology, 1995
    Co-Authors: George S Brush, Thomas J Kelly, Bruce Stillman
    Abstract:

    Publisher Summary At present, there is no in vitro DNA Replication assay suitable for the direct analysis of chromosomal DNA Replication in eukaryotes. Except in simple organisms such as yeast, the identification of Replication origins and the proteins that bind to these sites has been extremely difficult. Eukaryotic DNA Replication is tightly controlled during the cell cycle. It depends on the action of many regulatory proteins and to those directly involved in synthesizing DNA. One approach to circumvent these difficulties is to investigate the Replication of viral DNA within Eukaryotic cells. Viruses have small, well-defined genomes and the Replication of viral DNA is free of some of the normal cellular constraints. Simian virus 40 (SV40) DNA Replication requires only one viral protein; the large T antigen, which is an extremely useful tool for studying cellular DNA Replication. The development of an in vitro SV40 DNA Replication system I has led to the identification and characterization of many human Replication proteins and has allowed for the isolation of homologous proteins from other Eukaryotic species. This work has led to the realization that the basic Replication machinery is conserved in all eukaryotes from yeast to man. SV40 DNA Replication takes place within the nucleus of a permissive primate cell, where T antigen binds to and locally unwinds the circular SV40 minichromosome at the origin. This allows the host Replication proteins to initiate bidirectional DNA synthesis and complete the duplication of the entire DNA molecule in a semidiscontinuous manner. SV40 DNA Replication in vitro closely resembles the in vivo reaction and is dependent on the viral origin of Replication, T antigen, and primate cell cytoplasmic extract. The cellular components necessary and sufficient for the Replication of plasmid DNA containing the SV40 origin have been identified through fractionation of the cytoplasmic extract and reconstitution of activity.

Michael R Lieber - One of the best experts on this subject based on the ideXlab platform.

  • the fen 1 family of structure specific nucleases in Eukaryotic DNA Replication recombination and repair
    BioEssays, 1997
    Co-Authors: Michael R Lieber
    Abstract:

    Unlike the most well-characterized prokaryotic polymerase, E. Coli DNA pol I, none of the Eukaryotic polymerases have their own 5′ to 3′ exonuclease domain for nick translation and Okazaki fragment processing. In eukaryotes, FEN-1 is an endo-and exonuclease that carries out this function independently of the polymerase molecules. Only seven nucleases have been cloned from multicellular Eukaryotic cells. Among these, FEN-1 is intriguing because it has complex structural preferences; specifically, it cleaves at branched DNA structures. The cloning of FEN-1 permitted establishment of the first Eukaryotic nuclease family, predicting that S. cerevisiae RAD2 (S. pombe Rad13) and its mammalian homolog, XPG, would have similar structural specficity. The FEN-1 nuclease family includes several similar enzymes encoded by bacteriophages. The crystal structures of two enzymes in the FEN-1 nuclease family have been solved and they provide a structural basis for the interesting steric requirements of FEN-1 substrates. Because of their unique structural specificities, FEN-1 and its family members have important roles in DNA Replication, repair and, potentially, recombination. Recently, FEN-1 was found to specifically associate with PCNA, explaining some aspects of FEN-1 function during DNA Replication and potentially in DNA repair.

  • the fen 1 family of structure specific nucleases in Eukaryotic DNA Replication recombination and repair
    BioEssays, 1997
    Co-Authors: Michael R Lieber
    Abstract:

    Unlike the most well-characterized prokaryotic polymerase, E. Coli DNA pol I, none of the Eukaryotic polymerases have their own 5′ to 3′ exonuclease domain for nick translation and Okazaki fragment processing. In eukaryotes, FEN-1 is an endo-and exonuclease that carries out this function independently of the polymerase molecules. Only seven nucleases have been cloned from multicellular Eukaryotic cells. Among these, FEN-1 is intriguing because it has complex structural preferences; specifically, it cleaves at branched DNA structures. The cloning of FEN-1 permitted establishment of the first Eukaryotic nuclease family, predicting that S. cerevisiae RAD2 (S. pombe Rad13) and its mammalian homolog, XPG, would have similar structural specficity. The FEN-1 nuclease family includes several similar enzymes encoded by bacteriophages. The crystal structures of two enzymes in the FEN-1 nuclease family have been solved and they provide a structural basis for the interesting steric requirements of FEN-1 substrates. Because of their unique structural specificities, FEN-1 and its family members have important roles in DNA Replication, repair and, potentially, recombination. Recently, FEN-1 was found to specifically associate with PCNA, explaining some aspects of FEN-1 function during DNA Replication and potentially in DNA repair.

Joseph T P Yeeles - One of the best experts on this subject based on the ideXlab platform.

  • an updated perspective on the polymerase division of labor during Eukaryotic DNA Replication
    Critical Reviews in Biochemistry and Molecular Biology, 2020
    Co-Authors: Thomas A Guilliam, Joseph T P Yeeles
    Abstract:

    In eukaryotes three DNA polymerases (Pols), α, δ, and e, are tasked with bulk DNA synthesis of nascent strands during genome duplication. Most evidence supports a model where Pol α initiates DNA synthesis before Pol e and Pol δ replicate the leading and lagging strands, respectively. However, a number of recent reports, enabled by advances in biochemical and genetic techniques, have highlighted emerging roles for Pol δ in all stages of leading-strand synthesis; initiation, elongation, and termination, as well as fork restart. By focusing on these studies, this review provides an updated perspective on the division of labor between the replicative polymerases during DNA Replication.

  • reconstitution of translesion synthesis reveals a mechanism of Eukaryotic DNA Replication restart
    Nature Structural & Molecular Biology, 2020
    Co-Authors: Thomas A Guilliam, Joseph T P Yeeles
    Abstract:

    Leading-strand template aberrations cause helicase-polymerase uncoupling and impede Replication fork progression, but the details of how uncoupled forks are restarted remain uncertain. Using purified proteins from Saccharomyces cerevisiae, we have reconstituted translesion synthesis (TLS)-mediated restart of a Eukaryotic replisome following collision with a cyclobutane pyrimidine dimer. We find that TLS functions 'on the fly' to promote resumption of rapid Replication fork rates, despite lesion bypass occurring uncoupled from the Cdc45-MCM-GINS (CMG) helicase. Surprisingly, the main lagging-strand polymerase, Pol δ, binds the leading strand upon uncoupling and inhibits TLS. Pol δ is also crucial for efficient recoupling of leading-strand synthesis to CMG following lesion bypass. Proliferating cell nuclear antigen monoubiquitination positively regulates TLS to overcome Pol δ inhibition. We reveal that these mechanisms of negative and positive regulation also operate on the lagging strand. Our observations have implications for both fork restart and the division of labor during leading-strand synthesis generally.

  • mechanism of bidirectional leading strand synthesis establishment at Eukaryotic DNA Replication origins
    Molecular Cell, 2019
    Co-Authors: Valentina Aria, Joseph T P Yeeles
    Abstract:

    Summary DNA Replication commences at Eukaryotic Replication origins following assembly and activation of bidirectional CMG helicases. Once activated, CMG unwinds the parental DNA duplex and DNA polymerase α-primase initiates synthesis on both template strands. By utilizing an origin-dependent Replication system using purified yeast proteins, we have mapped start sites for leading-strand Replication. Synthesis is mostly initiated outside the origin sequence. Strikingly, rightward leading strands are primed left of the origin and vice versa. We show that each leading strand is established from a lagging-strand primer synthesized by the replisome on the opposite side of the origin. Preventing elongation of primers synthesized left of the origin blocked rightward leading strands, demonstrating that replisomes are interdependent for leading-strand synthesis establishment. The mechanism we reveal negates the need for dedicated leading-strand priming and necessitates a crucial role for the lagging-strand polymerase Pol δ in connecting the nascent leading strand with the advancing replisome.

  • regulated Eukaryotic DNA Replication origin firing with purified proteins
    Nature, 2015
    Co-Authors: Joseph T P Yeeles, Anne Early, Tom D Deegan, Agnieszka Janska, John F. X. Diffley
    Abstract:

    Eukaryotic cells initiate DNA Replication from multiple origins, which must be tightly regulated to promote precise genome duplication in every cell cycle. To accomplish this, initiation is partitioned into two temporally discrete steps: a double hexameric minichromosome maintenance (MCM) complex is first loaded at Replication origins during G1 phase, and then converted to the active CMG (Cdc45–MCM–GINS) helicase during S phase. Here we describe the reconstitution of budding yeast DNA Replication initiation with 16 purified Replication factors, made from 42 polypeptides. Origin-dependent initiation recapitulates regulation seen in vivo. Cyclin-dependent kinase (CDK) inhibits MCM loading by phosphorylating the origin recognition complex (ORC) and promotes CMG formation by phosphorylating Sld2 and Sld3. Dbf4-dependent kinase (DDK) promotes Replication by phosphorylating MCM, and can act either before or after CDK. These experiments define the minimum complement of proteins, protein kinase substrates and co-factors required for regulated Eukaryotic DNA Replication. It has long been a goal to reconstitute Eukaryotic DNA Replication; here a purified in vitro system from budding yeast containing 16 factors, themselves composed of 42 polypeptides, fulfils the staged process of origin-dependent initiation, including its regulation by kinases. It has been a long-desired goal to be able to reconstitute a Eukaryotic system of DNA Replication from its earliest stages of origin firing using purified proteins. However, the greater complexity of eukaryotes compared to bacterial and phage systems has hampered this development. But now John Diffley and colleagues have successfully reconstituted the initial events of budding yeast DNA Replication in vitro. The purified system contains 42 proteins, comprising 16 complexes, and fulfills the staged process of origin-dependent initiation, including its regulation by kinases.

  • regulated Eukaryotic DNA Replication origin firing with purified proteins
    Nature, 2015
    Co-Authors: Joseph T P Yeeles, Anne Early, Tom D Deegan, Agnieszka Janska, John F. X. Diffley
    Abstract:

    Eukaryotic cells initiate DNA Replication from multiple origins, which must be tightly regulated to promote precise genome duplication in every cell cycle. To accomplish this, initiation is partitioned into two temporally discrete steps: a double hexameric minichromosome maintenance (MCM) complex is first loaded at Replication origins during G1 phase, and then converted to the active CMG (Cdc45-MCM-GINS) helicase during S phase. Here we describe the reconstitution of budding yeast DNA Replication initiation with 16 purified Replication factors, made from 42 polypeptides. Origin-dependent initiation recapitulates regulation seen in vivo. Cyclin-dependent kinase (CDK) inhibits MCM loading by phosphorylating the origin recognition complex (ORC) and promotes CMG formation by phosphorylating Sld2 and Sld3. Dbf4-dependent kinase (DDK) promotes Replication by phosphorylating MCM, and can act either before or after CDK. These experiments define the minimum complement of proteins, protein kinase substrates and co-factors required for regulated Eukaryotic DNA Replication.

Johannes C. Walter - One of the best experts on this subject based on the ideXlab platform.

  • uncoupling of sister replisomes during Eukaryotic DNA Replication
    Molecular Cell, 2010
    Co-Authors: Hasan Yardimci, Anna B Loveland, Satoshi Habuchi, Antoine M Van Oijen, Johannes C. Walter
    Abstract:

    The duplication of Eukaryotic genomes involves the Replication of DNA from multiple origins of Replication. In S phase, two sister replisomes assemble at each active origin, and they replicate DNA in opposite directions. Little is known about the functional relationship between sister replisomes. Some data imply that they travel away from one another and thus function independently. Alternatively, sister replisomes may form a stationary, functional unit that draws parental DNA toward itself. If this "double replisome" model is correct, a constrained DNA molecule should not undergo Replication. To test this prediction, lambda DNA was stretched and immobilized at both ends within a microfluidic flow cell. Upon exposure to Xenopus egg extracts, this DNA underwent extensive Replication by a single pair of diverging replisomes. The data show that there is no obligatory coupling between sister replisomes and, together with other studies, imply that genome duplication involves autonomously functioning replisomes.

  • localization of mcm2 7 cdc45 and gins to the site of DNA unwinding during Eukaryotic DNA Replication
    Molecular Cell, 2006
    Co-Authors: Marcin Pacek, Haruhiko Takisawa, Yumiko Kubota, Antonin V Tutter, Johannes C. Walter
    Abstract:

    Summary Little is known about the architecture and biochemical composition of the Eukaryotic DNA Replication fork. To study this problem, we used biotin-streptavidin-modified plasmids to induce sequence-specific Replication fork pausing in Xenopus egg extracts. Chromatin immunoprecipitation was employed to identify factors associated with the paused fork. This approach identifies DNA pol α, DNA pol δ, DNA pol ɛ, MCM2-7, Cdc45, GINS, and Mcm10 as components of the vertebrate replisome. In the presence of the DNA polymerase inhibitor aphidicolin, which causes uncoupling of a highly processive DNA helicase from the stalled replisome, only Cdc45, GINS, and MCM2-7 are enriched at the pause site. The data suggest the existence of a large molecular machine, the "unwindosome," which separates DNA strands at the Replication fork and contains Cdc45, GINS, and the MCM2-7 holocomplex.

  • Eukaryotic origins of DNA Replication could you please be more specific
    Seminars in Cell & Developmental Biology, 2005
    Co-Authors: Christin Cvetic, Johannes C. Walter
    Abstract:

    Initiation of Eukaryotic DNA Replication commences when the origin recognition complex (ORC) binds to DNA, recruiting helicases, polymerases, and necessary cofactors. While the biochemical mechanism and factors involved in Replication initiation appear to be highly conserved, the DNA sequences at which these events take place in different organisms are not. Thus, while ORC appears to bind to specific DNA sequences in budding yeast, there is increasing new evidence that metazoan ORC complexes do not rely on sequence to be directed to origins of Replication. Here, we review examples of specific and non-specific initiation, and we consider what, if not DNA sequence, accounts for DNA binding of ORC to defined regions in Eukaryotic genomes.

  • A requirement for MCM7 and Cdc45 in chromosome unwinding during Eukaryotic DNA Replication.
    The EMBO Journal, 2004
    Co-Authors: Marcin Pacek, Johannes C. Walter
    Abstract:

    In vertebrates, MCM2–7 and Cdc45 are required for DNA Replication initiation, but it is unknown whether they are also required for elongation, as in yeast. Moreover, although MCM2–7 is a prime candidate for the Eukaryotic replicative DNA helicase, a demonstration that MCM2–7 unwinds DNA during Replication is lacking. Here, we use Xenopus egg extracts to investigate the roles of MCM7 and Cdc45 in DNA Replication. A fragment of the retinoblastoma protein, Rb1−400, was used to neutralize MCM7, and antibodies were used to neutralize Cdc45. When added immediately after origin unwinding, or after significant DNA synthesis, both inhibitors blocked further DNA Replication, indicating that MCM7 and Cdc45 are required throughout Replication elongation in vertebrates. We next exploited the fact that inhibition of DNA polymerase by aphidicolin causes extensive chromosome unwinding, likely due to uncoupling of the replicative DNA helicase. Strikingly, Rb1−400 and Cdc45 antibodies both abolished unwinding by the uncoupled helicase. These results provide new support for the model that MCM2–7 is the replicative DNA helicase, and they indicate that Cdc45 functions as a helicase co-factor.

  • inhibition of Eukaryotic DNA Replication by geminin binding to cdt1
    Science, 2000
    Co-Authors: James A Wohlschlegel, Johannes C. Walter, Brian T Dwyer, Suman Kumar Dhar, Christin Cvetic, Anindya Dutta
    Abstract:

    In all Eukaryotic organisms, inappropriate firing of Replication origins during the G2 phase of the cell cycle is suppressed by cyclin-dependent kinases. Multicellular eukaryotes contain a second putative inhibitor of re-Replication called geminin. Geminin is believed to block binding of the mini-chromosome maintenance (MCM) complex to origins of Replication, but the mechanism of this inhibition is unclear. Here we show that geminin interacts tightly with Cdt1, a recently identified Replication initiation factor necessary for MCM loading. The inhibition of DNA Replication by geminin that is observed in cell-free DNA Replication extracts is reversed by the addition of excess Cdt1. In the normal cell cycle, Cdt1 is present only in G1 and S, whereas geminin is present in S and G2 phases of the cell cycle. Together, these results suggest that geminin inhibits inappropriate origin firing by targeting Cdt1.