The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform

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

  • the dynamic nature of the human Origin Recognition Complex revealed through five cryoem structures
    eLife, 2020
    Co-Authors: Bruce Stillman, Matt J Jaremko, Dennis R Thomas, Leemor Joshuator
    Abstract:

    Genome replication is initiated from specific Origin sites established by dynamic events. The Origin Recognition Complex (ORC) is necessary for orchestrating the initiation process by binding to Origin DNA, recruiting CDC6, and assembling the MCM replicative helicase on DNA. Here we report five cryoEM structures of the human ORC (HsORC) that illustrate the native flexibility of the Complex. The absence of ORC1 revealed a compact, stable Complex of ORC2-5. Introduction of ORC1 opens the Complex into several dynamic conformations. Two structures revealed dynamic movements of the ORC1 AAA+ and ORC2 winged-helix domains that likely impact DNA incorporation into the ORC core. Additional twist and pinch motions were observed in an open ORC conformation revealing a hinge at the ORC5·ORC3 interface that may facilitate ORC binding to DNA. Finally, a structure of ORC was determined with endogenous DNA bound in the core revealing important differences between human and yeast Origin Recognition.

  • structure of the active form of human Origin Recognition Complex and its atpase motor module
    eLife, 2017
    Co-Authors: Zuanning Yuan, Ante Tocilj, Jingchuan Sun, Elad Elkayam, Bruce Stillman
    Abstract:

    Binding of the Origin Recognition Complex (ORC) to Origins of replication marks the first step in the initiation of replication of the genome in all eukaryotic cells. Here, we report the structure of the active form of human ORC determined by X-ray crystallography and cryo-electron microscopy. The Complex is composed of an ORC1/4/5 motor module lobe in an organization reminiscent of the DNA polymerase clamp loader Complexes. A second lobe contains the ORC2/3 subunits. The Complex is organized as a double-layered shallow corkscrew, with the AAA+ and AAA+-like domains forming one layer, and the winged-helix domains (WHDs) forming a top layer. CDC6 fits easily between ORC1 and ORC2, completing the ring and the DNA-binding channel, forming an additional ATP hydrolysis site. Analysis of the ATPase activity of the Complex provides a basis for understanding ORC activity as well as molecular defects observed in Meier-Gorlin Syndrome mutations.

  • orc1 binding to mitotic chromosomes precedes spatial patterning during g1 phase and assembly of the Origin Recognition Complex in human cells
    Journal of Biological Chemistry, 2015
    Co-Authors: Nihan Kara, Manzar Hossain, Supriya G Prasanth, Bruce Stillman
    Abstract:

    Replication of eukaryotic chromosomes occurs once every cell division cycle in normal cells and is a tightly controlled process that ensures complete genome duplication. The Origin Recognition Complex (ORC) plays a key role during the initiation of DNA replication. In human cells, the level of Orc1, the largest subunit of ORC, is regulated during the cell division cycle, and thus ORC is a dynamic Complex. Upon S phase entry, Orc1 is ubiquitinated and targeted for destruction, with subsequent dissociation of ORC from chromosomes. Time lapse and live cell images of human cells expressing fluorescently tagged Orc1 show that Orc1 re-localizes to condensing chromatin during early mitosis and then displays different nuclear localization patterns at different times during G1 phase, remaining associated with late replicating regions of the genome in late G1 phase. The initial binding of Orc1 to mitotic chromosomes requires C-terminal amino acid sequences that are similar to mitotic chromosome-binding sequences in the transcriptional pioneer protein FOXA1. Depletion of Orc1 causes concomitant loss of the mini-chromosome maintenance (Mcm2–7) helicase proteins on chromatin. The data suggest that Orc1 acts as a nucleating center for ORC assembly and then pre-replication Complex assembly by binding to mitotic chromosomes, followed by gradual removal from chromatin during the G1 phase. Background: Orc1 is the largest subunit of the Origin Recognition Complex that promotes genome duplication. Results: We studied the dynamics of Orc1 during the cell division cycle. Conclusion: Orc1 binds to mitotic chromosomes, and during G1 phase in the daughter cells it then forms spatial-temporal patterns in the nucleus. Significance: The large subunit of ORC orchestrates the earliest stages of chromosome inheritance.

  • the Origin Recognition Complex a biochemical and structural view
    Sub-cellular biochemistry, 2012
    Co-Authors: Bruce Stillman
    Abstract:

    The Origin Recognition Complex (ORC) was first discovered in the baker's yeast in 1992. Identification of ORC opened up a path for subsequent molecular level investigations on how eukaryotic cells initiate and control genome duplication each cell cycle. Twenty years after the first biochemical isolation, ORC is now taking on a three-dimensional shape, although a very blurry shape at the moment, thanks to the recent electron microscopy and image reconstruction efforts. In this chapter, we outline the current biochemical knowledge about ORC from several eukaryotic systems, with emphasis on the most recent structural and biochemical studies. Despite many species-specific properties, an emerging consensus is that ORC is an ATP-dependent machine that recruits other key proteins to form pre-replicative Complexes (pre-RCs) at many Origins of DNA replication, enabling the subsequent initiation of DNA replication in S phase.

  • human Origin Recognition Complex is essential for hp1 binding to chromatin and heterochromatin organization
    Proceedings of the National Academy of Sciences of the United States of America, 2010
    Co-Authors: Supriya G Prasanth, Zhen Shen, Kannanganattu V Prasanth, Bruce Stillman
    Abstract:

    The Origin Recognition Complex (ORC) is a DNA replication initiator protein also known to be involved in diverse cellular functions including gene silencing, sister chromatid cohesion, telomere biology, heterochromatin localization, centromere and centrosome activity, and cytokinesis. We show that, in human cells, multiple ORC subunits associate with hetereochromatin protein 1 (HP1) alpha- and HP1beta-containing heterochromatic foci. Fluorescent bleaching studies indicate that multiple subComplexes of ORC exist at heterochromatin, with Orc1 stably associating with heterochromatin in G1 phase, whereas other ORC subunits have transient interactions throughout the cell-division cycle. Both Orc1 and Orc3 directly bind to HP1alpha, and two domains of Orc3, a coiled-coil domain and a mod-interacting region domain, can independently bind to HP1alpha; however, both are essential for in vivo localization of Orc3 to heterochromatic foci. Direct binding of both Orc1 and Orc3 to HP1 suggests that, after the degradation of Orc1 at the G1/S boundary, Orc3 facilitates assembly of ORC/HP1 proteins to chromatin. Although depletion of Orc2 and Orc3 subunits by siRNA caused loss of HP1alpha association to heterochromatin, loss of Orc1 and Orc5 caused aberrant HP1alpha distribution only to pericentric heterochromatin-surrounding nucleoli. Depletion of HP1alpha from human cells also shows loss of Orc2 binding to heterochromatin, suggesting that ORC and HP1 proteins are mutually required for each other to bind to heterochromatin. Similar to HP1alpha-depleted cells, Orc2 and Orc3 siRNA-treated cells also show loss of compaction at satellite repeats, suggesting that ORC together with HP1 proteins may be involved in organizing higher-order chromatin structure and centromere function.

Jasper Rine - One of the best experts on this subject based on the ideXlab platform.

  • expanded roles of the Origin Recognition Complex in the architecture and function of silenced chromatin in saccharomyces cerevisiae
    Molecular and Cellular Biology, 2010
    Co-Authors: Bilge Ozaydin, Jasper Rine
    Abstract:

    The silenced chromatin at the cryptic mating-type loci (HML and HMR) of Saccharomyces cerevisiae requires a cell cycle event between early S phase and G(2)/M phase to achieve repression. Although DNA replication per se is not essential for silencing, mutations in many of the proteins involved in DNA replication affect silencing. Each of the four silencers, which flank the silenced loci, includes an Origin Recognition Complex (ORC) binding site (ACS). ORC directly interacted with Sir1 and recruits Sir1 to the silencers. This study describes additional roles for ORC in the architecture of silenced chromatin. Using chromatin immunoprecipitation (ChIP) analysis, we found that ORC physically interacts throughout the internal regions of HMR as well as with silencers. This interaction depended on the presence of Sir proteins and, in part, on the HMR-I silencer. ORC remained associated with the internal regions of HMR even when these regions were recombinationally separated from the silencers. Moreover, ORC could be recruited to the silencers lacking an ACS through its Sir1 interaction.

  • association with the Origin Recognition Complex suggests a novel role for histone acetyltransferase hat1p hat2p
    BMC Biology, 2007
    Co-Authors: Jasper Rine, Bernhard Suter, Oxana Pogoutse, Xinghua Guo, Nevan J Krogan, Peter N Lewis, Jack Greenblatt, Andrew Emili
    Abstract:

    Histone modifications have been implicated in the regulation of transcription and, more recently, in DNA replication and repair. In yeast, a major conserved histone acetyltransferase, Hat1p, preferentially acetylates lysine residues 5 and 12 on histone H4. Here, we report that a nuclear sub-Complex consisting of Hat1p and its partner Hat2p interacts physically and functionally with the Origin Recognition Complex (ORC). While mutational inactivation of the histone acetyltransferase (HAT) gene HAT1 alone does not compromise Origin firing or initiation of DNA replication, a deletion in HAT1 (or HAT2) exacerbates the growth defects of conditional orc-ts mutants. Thus, the ORC-associated Hat1p-dependent histone acetyltransferase activity suggests a novel linkage between histone modification and DNA replication. Additional genetic and biochemical evidence points to the existence of partly overlapping histone H3 acetyltransferase activities in addition to Hat1p/Hat2p for proper DNA replication efficiency. Furthermore, we demonstrated a dynamic association of Hat1p with chromatin during S-phase that suggests a role of this enzyme at the replication fork. We have found an intriguing new association of the Hat1p-dependent histone acetyltransferase in addition to its previously known role in nuclear chromatin assembly (Hat1p/Hat2p-Hif1p). The participation of a distinct Hat1p/Hat2p sub-Complex suggests a linkage of histone H4 modification with ORC-dependent DNA replication.

  • sum1p the Origin Recognition Complex and the spreading of a promoter specific repressor in saccharomyces cerevisiae
    Molecular and Cellular Biology, 2005
    Co-Authors: Patrick J Lynch, Jasper Rine, Hunter B Fraser, Elena Sevastopoulos, Laura N Rusche
    Abstract:

    In Saccharomyces cerevisiae, Sum1p is a promoter-specific repressor. A single amino acid change generates the mutant Sum1-1p, which causes regional silencing at new loci where wild-type Sum1p does not act. Thus, Sum1-1p is a model for understanding how the spreading of repressive chromatin is regulated. When wild-type Sum1p was targeted to a locus where mutant Sum1-1p spreads, wild-type Sum1p did not spread as efficiently as mutant Sum1-1p did, despite being in the same genomic context. Thus, the SUM1-1 mutation altered the ability of the protein to spread. The spreading of Sum1-1p required both an enzymatically active deacetylase, Hst1p, and the N-terminal tail of histone H4, consistent with the spreading of Sum1-1p involving sequential modification of and binding to histone tails, as observed for other silencing proteins. Furthermore, deletion of the N-terminal tail of H4 caused Sum1-1p to return to loci where wild-type Sum1p acts, consistent with the SUM1-1 mutation increasing the affinity of the protein for H4 tails. These results imply that the spreading of repressive chromatin proteins is regulated by their affinities for histone tails. Finally, this study uncovered a functional connection between wild-type Sum1p and the Origin Recognition Complex, and this relationship also contributes to mutant Sum1-1p localization.

  • the Origin Recognition Complex links replication sister chromatid cohesion and transcriptional silencing in saccharomyces cerevisiae
    Genetics, 2004
    Co-Authors: Bernhard Suter, Amy Hin Yan Tong, Michael Chang, Grant W Brown, Charles Boone, Jasper Rine
    Abstract:

    Mutations in genes encoding the Origin Recognition Complex (ORC) of Saccharomyces cerevisiae affect initiation of DNA replication and transcriptional repression at the silent mating-type loci. To explore the function of ORC in more detail, a screen for genetic interactions was undertaken using large-scale synthetic lethal analysis. Combination of orc2-1 and orc5-1 alleles with the complete set of haploid deletion mutants revealed synthetic lethal/sick phenotypes with genes involved in DNA replication, chromatin structure, checkpoints, DNA repair and recombination, and other genes that were unexpected on the basis of previous studies of ORC. Many of these genetic interactions are shared with other genes that are involved in initiation of DNA replication. Strong synthetic interactions were demonstrated with null mutations in genes that contribute to sister chromatid cohesion. A genetic interaction between orc5-1 and the cohesin mutant scc1-73 suggested that ORC function contributes to sister chromatid cohesion. Thus, comprehensive screening for genetic interactions with a replication gene revealed a connection between initiation of DNA replication and sister chromatid cohesion. Further experiments linked sister chromatid cohesion genes to silencing at mating-type loci and telomeres.

  • the Origin Recognition Complex sir1 and the s phase requirement for silencing
    Science, 1997
    Co-Authors: Catherine A Fox, Ann E Ehrenhofermurray, Stephen Loo, Jasper Rine
    Abstract:

    Silencing of transcription in Saccharomyces cerevisiae has several links to DNA replication, including a role for the Origin Recognition Complex (ORC), the DNA replication initiator, in both processes. In addition, the establishment of silencing at the HML and HMR loci requires cells to pass through the S phase of the cell cycle. Passage through S phase was required for silencing of HMR even under conditions in which ORC itself was no longer required. The requirement for ORC in silencing of HMR could be bypassed by tethering the Sir1 protein to the HMR-E silencer. However, ORC had a Sir1-independent role in transcriptional silencing at telomeres. Thus, the role of ORC in silencing was separable from its role in initiation, and the role of S phase in silencing was independent of replication initiation at the silencers.

Stephen P Bell - One of the best experts on this subject based on the ideXlab platform.

  • mapping subunit location on the saccharomyces cerevisiae Origin Recognition Complex free and bound to dna using a novel nanoscale biopointer
    Journal of Biological Chemistry, 2004
    Co-Authors: Paul D Chastain, Stephen P Bell, Jayson L Bowers, Daniel G Lee, Jack D Griffith
    Abstract:

    The Saccharomyces cerevisiae Origin Recognition Complex (ORC) is composed of six subunits and is an essential component in the assembly of the replication apparatus. To probe the organization of this multiprotein Complex by electron microscopy, each subunit was tagged on either its C or N terminus with biotin and assembled into a Complex with the five other unmodified subunits. A nanoscale biopointer consisting of a short DNA duplex with streptavidin at one end was used to map the location of the N and C termini of each subunit. These observations were made using ORC free in solution and bound to the ARS1 Origin of replication. This mapping confirms and extends previous studies mapping the sites of subunit interaction with Origin DNA. In particular, we provide new information concerning the stoichiometry of the ORC-ARS1 Complex and the changes in conformation that are associated with DNA binding by ORC. This versatile, new approach to mapping protein structure has potential for many applications.

  • atp bound to the Origin Recognition Complex is important for prerc formation
    Proceedings of the National Academy of Sciences of the United States of America, 2001
    Co-Authors: Richard D Klemm, Stephen P Bell
    Abstract:

    The Origin Recognition Complex (ORC) binds Origins of replication and directs the assembly of a higher order protein Complex at these sites. ORC binds and hydrolyzes ATP in vitro. ATP binding to the largest subunit of ORC, Orc1p, stimulates specific binding to Origin DNA; however, the function of ATP hydrolysis by ORC is unknown. To address the role of ATP hydrolysis, we have generated mutants within Orc1p that are dominant lethal. At physiological ATP concentrations, these mutants are defective for ATP hydrolysis but not ATP binding in the absence of DNA. These mutants inhibit formation of the prereplicative Complex when overexpressed. The dominant lethal phenotype of these mutant ORC Complexes is suppressed by simultaneous overexpression of wild-type, but not mutant, Cdc6p. Our findings suggest that these hydrolysis-defective mutants inhibit growth by titrating Cdc6p away from the Origin. Based on these observations, we propose that Cdc6p specifically recognizes the ATP-bound state of Orc1p and that ATP hydrolysis is coupled to preRC disassembly.

  • regulation of Origin Recognition Complex conformation and atpase activity differential effects of single stranded and double stranded dna binding
    The EMBO Journal, 2000
    Co-Authors: Daniel G Lee, Richard D Klemm, Jack D Griffith, Alexander M Makhov, Stephen P Bell
    Abstract:

    The Saccharomyces cerevisiae Origin Recognition Complex (ORC) is bound to Origins of DNA replication throughout the cell cycle and directs the assembly of higher-order protein–DNA Complexes during G1. To examine the fate of ORC when Origin DNA is unwound during replication initiation, we determined the effect of single-stranded DNA (ssDNA) on ORC. We show that ORC can bind ssDNA and that ORC bound to ssDNA is distinct from that bound to double-stranded Origin DNA. ssDNA stimulated ORC ATPase activity, whereas double-stranded Origin DNA inhibited the same activity. Electron microscopy studies revealed two alternative conformations of ORC: an extended conformation stabilized by Origin DNA and a bent conformation stabilized by ssDNA. Therefore, ORC appears to exist in two distinct states with respect to its conformation and ATPase activity. Interestingly, the effect of ssDNA on these properties of ORC is correlated with ssDNA length. Since double-stranded Origin DNA and ssDNA differentially stabilize these two forms of ORC, we propose that Origin unwinding triggers a transition between these alternative states.

  • architecture of the yeast Origin Recognition Complex bound to Origins of dna replication
    Molecular and Cellular Biology, 1997
    Co-Authors: Stephen P Bell
    Abstract:

    : In many organisms, the replication of DNA requires the binding of a protein called the initiator to DNA sites referred to as Origins of replication. Analyses of multiple initiator proteins bound to their cognate Origins have provided important insights into the mechanism by which DNA replication is initiated. To extend this level of analysis to the study of eukaryotic chromosomal replication, we have investigated the architecture of the Saccharomyces cerevisiae Origin Recognition Complex (ORC) bound to yeast Origins of replication. Determination of DNA residues important for ORC-Origin association indicated that ORC interacts preferentially with one strand of the ARS1 Origin of replication. DNA binding assays using ORC Complexes lacking one of the six subunits demonstrated that the DNA binding domain of ORC requires the coordinate action of five of the six ORC subunits. Protein-DNA cross-linking studies suggested that Recognition of Origin sequences is mediated primarily by two different groups of ORC subunits that make sequence-specific contacts with two distinct regions of the DNA. Implications of these findings for ORC function and the mechanism of initiation of eukaryotic DNA replication are discussed.

  • coordinate binding of atp and Origin dna regulates the atpase activity of the Origin Recognition Complex
    Cell, 1997
    Co-Authors: Richard D Klemm, Richard J Austin, Stephen P Bell
    Abstract:

    Abstract The Origin Recognition Complex (ORC) is a six-protein assembly that specifies the sites of DNA replication initiation in S. cerevisiae. Origin Recognition by ORC requires ATP. Here, we demonstrate that two subunits, Orc1p and Orc5p, bind ATP and that Orc1p also hydrolyzes ATP. ATP binding and hydrolysis by Orc1p are both regulated by Origin DNA in a sequence-specific manner. ATP binding to Orc1p, but not ATP hydrolysis, is responsible for the ATP dependence of the ORC–Origin interaction, indicating that ATP is a cofactor that locks ORC on Origin DNA. These data demonstrate that occupancy of the Orc1p ATP–binding site has a profound effect on ORC function and that ATP hydrolysis by Orc1p has the potential to drive transitions between different functional states of ORC.

Susan M Gasser - One of the best experts on this subject based on the ideXlab platform.

  • the Origin Recognition Complex functions in sister chromatid cohesion in saccharomyces cerevisiae
    Cell, 2007
    Co-Authors: Kenji Shimada, Susan M Gasser
    Abstract:

    High-fidelity chromosomal segregation requires the properly timed establishment of sister-chromatid cohesion mediated by the Cohesin Complex, and its resolution at the metaphase-to-anaphase transition. We have examined cell-cycle progression in a yeast strain from which the Origin Recognition Complex protein Orc2 was depleted after the assembly of prereplication Complexes. We find that Orc2 depletion causes a delay in progression through mitosis, reflecting activation of both the DNA-damage and Mad2-spindle checkpoints. Surprisingly, sister-chromatid cohesion is impaired in Orc2-depleted cells, although Cohesin subunits are properly associated with chromatin. Reexpression of Orc2 in late G2/M phase restores chromatid cohesion. Finally, the targeting of Orc2 to a specific chromosomal locus suppresses premature sister-chromatid separation locally in a temperature-sensitive cohesin mutant. We conclude that ORC mediates sister-chromatid interaction on a pathway that is additive with Cohesin-mediated pairing.

  • an n terminal domain of dbf4p mediates interaction with both Origin Recognition Complex orc and rad53p and can deregulate late Origin firing
    Proceedings of the National Academy of Sciences of the United States of America, 2002
    Co-Authors: Bernard P Duncker, Kenji Shimada, Monika Tsaipflugfelder, Philippe Pasero, Susan M Gasser
    Abstract:

    The Dbf4/Cdc7 kinase acts at the level of individual Origins to promote the initiation of DNA replication. We demonstrate through both immunoprecipitation and two-hybrid assays that a domain comprising the first 296 aa of Dbf4p interacts with Orc2p and Orc3p subunits of the Origin Recognition Complex (ORC). Given that the activation of Rad53 kinase in response to the DNA replication checkpoint leads to the release of Dbf4p from an ORC-containing chromatin fraction, we also examined interaction between Dbf4p and Rad53p. This same domain of Dbf4p binds specifically to the forkhead homology-associated (FHA) domains of Rad53p. Cell cycle arrest in G2/M, provoked by the overexpression of the Dbf4 domain, is suppressed in a rad53 mutant. Moreover, its overexpression perturbs the regulation of late, but not early, Origin firing in wild-type cells after treatment with hydroxyurea.

Thomas J Kelly - One of the best experts on this subject based on the ideXlab platform.

  • multiple mechanisms contribute to schizosaccharomyces pombe Origin Recognition Complex dna interactions
    Journal of Biological Chemistry, 2008
    Co-Authors: Christopher Houchens, Rayyuan Chuang, Mark G Frattini, Alex Fuller, Pam Simancek, Thomas J Kelly
    Abstract:

    Eukaryotic DNA replication requires the assembly of multiprotein pre-replication Complexes (pre-RCs) at chromosomal Origins of DNA replication. Here we describe the interactions of highly purified Schizosaccharomyces pombe pre-RC components, SpORC, SpCdc18, and SpCdt1, with each other and with ars1 Origin DNA. We show that SpORC binds DNA in at least two steps. The first step likely involves electrostatic interactions between the AT-hook motifs of SpOrc4 and AT tracts in ars1 DNA and results in the formation of a salt-sensitive Complex. In the second step, the salt-sensitive Complex is slowly converted to a salt-stable Complex that involves additional interactions between SpORC and DNA. Binding of SpORC to ars1 DNA is facilitated by negative supercoiling and is accompanied by changes in DNA topology, suggesting that SpORC-DNA Complexes contain underwound or negatively writhed DNA. Purified human Origin Recognition Complex (ORC) induces similar topological changes in Origin DNA, indicating that this property of ORC is conserved in eukaryotic evolution and plays an important role in ORC function. We also show that SpCdc18 and SpCdt1 form a binary Complex that has greater affinity for DNA than either protein alone. In addition, both proteins contribute significantly to the stability of the initial SpORC-DNA Complex and enhance the SpORC-dependent topology changes in Origin DNA. Thus, the formation of stable protein-DNA Complexes at S. pombe Origins of replication involves binary interactions among all three proteins, as well as interactions of both SpORC and SpCdt1-SpCdc18 with Origin DNA. These findings demonstrate that SpORC is not the sole determinant of Origin Recognition.

  • sequence independent dna binding and replication initiation by the human Origin Recognition Complex
    Genes & Development, 2003
    Co-Authors: Sanjay Vashee, Pamela Simancek, Thomas J Kelly, Christin Cvetic, Wenyan Lu, Johannes C Walter
    Abstract:

    We report that a highly purified human Origin Recognition Complex (HsORC) has intrinsic DNA-binding activity, and that this activity is modestly stimulated by ATP. HsORC binds preferentially to synthetic AT-rich polydeoxynucleotides, but does not effectively discriminate between natural DNA fragments that contain known human Origins and control fragments. The Complex fully restores DNA replication to ORC-depleted Xenopus egg extracts, providing strong evidence for its initiator function. Strikingly, HsORC stimulates initiation from any DNA sequence, and it does not preferentially replicate DNA containing human Origin sequences. These data provide a biochemical explanation for the observation that in metazoans, initiation of DNA replication often occurs in a seemingly random pattern, and they have important implications for the nature of human Origins of DNA replication.

  • purification and characterization of the schizosaccharomyces pombe Origin Recognition Complex interaction with Origin dna and cdc18 protein
    Journal of Biological Chemistry, 2002
    Co-Authors: Rayyuan Chuang, Louise Chretien, Jianli Dai, Thomas J Kelly
    Abstract:

    The Origin Recognition Complex (ORC) plays a central role in the initiation of DNA replication in eukaryotic cells. It interacts with Origins of DNA replication in chromosomal DNA and recruits additional replication proteins to form functional initiation Complexes. These processes have not been well characterized at the biochemical level except in the case of Saccharomyces cerevisiae ORC. We report here the expression, purification, and initial characterization of Schizosaccharomyces pombeORC (SpORC) containing six recombinant subunits. Purified SpORC binds efficiently to the ars1 Origin of DNA replication via the essential Nterminal domain of the SpOrc4 subunit which contains nine AT-hook motifs. Competition binding experiments demonstrated that SpORC binds preferentially to DNA molecules rich in AT-tracts, but does not otherwise exhibit a high degree of sequence specificity. The Complex is capable of binding to multiple sites within the ars1 Origin of DNA replication with similar affinities, indicating that the sequence requirements for Origin Recognition in S. pombe are significantly less stringent than in S. cerevisiae. We have also demonstrated that SpORC interacts directly with Cdc18p, an essential fission yeast initiation protein, and recruits it to the ars1 Origin in vitro. Recruitment of Cdc18p to chromosomal Origins is a likely early step in the initiation of DNA replication in vivo. These data indicate that the purified recombinant SpORC retains at least two of its primary biological functions and that it will be useful for the eventual reconstitution of the initiation reaction with purified proteins.

  • assembly of the human Origin Recognition Complex
    Journal of Biological Chemistry, 2001
    Co-Authors: Sanjay Vashee, Pamela Simancek, Mark D Challberg, Thomas J Kelly
    Abstract:

    The six-subunit Origin Recognition Complex (ORC) was Originally identified in the yeast Saccharomyces cerevisiae. Yeast ORC binds specifically to Origins of replication and serves as a platform for the assembly of additional initiation factors, such as Cdc6 and the Mcm proteins. Human homologues of all six ORC subunits have been identified by sequence similarity to their yeast counterparts, but little is known about the biochemical characteristics of human ORC (HsORC). We have extracted HsORC from HeLa cell chromatin and probed its subunit composition using specific antibodies. The endogenous HsORC, identified in these experiments, contained homologues of Orc1-Orc5 but lacked a putative homologue of Orc6. By expressing HsORC subunits in insect cells using the baculovirus system, we were able to identify a Complex containing all six subunits. To explore the subunit-subunit interactions that are required for the assembly of HsORC, we carried out extensive co-immunoprecipitation experiments with recombinant ORC subunits expressed in different combinations. These studies revealed the following binary interactions: HsOrc2-HsOrc3, HsOrc2-HsOrc4, HsOrc3-HsOrc4, HsOrc2-HsOrc6, and HsOrc3-HsOrc6. HsOrc5 did not form stable binary Complexes with any other HsORC subunit but interacted with sub-Complexes containing any two of subunits HsOrc2, HsOrc3, or HsOrc4. Complex formation by HsOrc1 required the presence of HsOrc2, HsOrc3, HsOrc4, and HsOrc5 subunits. These results suggest that the subunits HsOrc2, HsOrc3, and HsOrc4 form a core upon which the ordered assembly of HsOrc5 and HsOrc1 takes place. The characterization of HsORC should facilitate the identification of human Origins of DNA replication.