scholarly journals The dynamic nature of the human origin recognition complex revealed through five cryoEM structures

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Matt J Jaremko ◽  
Kin Fan On ◽  
Dennis R Thomas ◽  
Bruce Stillman ◽  
Leemor Joshua-Tor
Keyword(s):  
Origin Recognition Complex ◽  
Stable Complex ◽  
Genome Replication ◽  
Dynamic Nature ◽  
Winged Helix ◽  
Replicative Helicase ◽  
The Core ◽  
Dynamic Events ◽  
Dna Incorporation ◽  
Origin Recognition

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.

scholarly journals The dynamic nature of the human Origin Recognition Complex revealed through five cryoEM structures

2020 ◽  
Author(s):  
Matt J. Jaremko ◽  
Kin Fan On ◽  
Dennis R. Thomas ◽  
Bruce Stillman ◽  
Leemor Joshua-Tor
Keyword(s):  
Origin Recognition Complex ◽  
Stable Complex ◽  
Genome Replication ◽  
Dynamic Nature ◽  
Winged Helix ◽  
Replicative Helicase ◽  
The Core ◽  
Dynamic Events ◽  
Dna Incorporation ◽  
Origin Recognition

AbstractGenome 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 ORC5·3 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.

scholarly journals The structure of ORC–Cdc6 on an origin DNA reveals the mechanism of ORC activation by the replication initiator Cdc6

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Xiang Feng ◽  
Yasunori Noguchi ◽  
Marta Barbon ◽  
Bruce Stillman ◽  
Christian Speck ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Origin Recognition Complex ◽  
Molecular Level ◽  
Dna Recognition ◽  
Winged Helix ◽  
Replicative Helicase ◽  
The Core ◽  
Α Helix ◽  
Replication Initiator ◽  
Winged Helix Domain

AbstractThe Origin Recognition Complex (ORC) binds to sites in chromosomes to specify the location of origins of DNA replication. The S. cerevisiae ORC binds to specific DNA sequences throughout the cell cycle but becomes active only when it binds to the replication initiator Cdc6. It has been unclear at the molecular level how Cdc6 activates ORC, converting it to an active recruiter of the Mcm2-7 hexamer, the core of the replicative helicase. Here we report the cryo-EM structure at 3.3 Å resolution of the yeast ORC–Cdc6 bound to an 85-bp ARS1 origin DNA. The structure reveals that Cdc6 contributes to origin DNA recognition via its winged helix domain (WHD) and its initiator-specific motif. Cdc6 binding rearranges a short α-helix in the Orc1 AAA+ domain and the Orc2 WHD, leading to the activation of the Cdc6 ATPase and the formation of the three sites for the recruitment of Mcm2-7, none of which are present in ORC alone. The results illuminate the molecular mechanism of a critical biochemical step in the licensing of eukaryotic replication origins.

scholarly journals Conformational control and DNA-binding mechanism of the metazoan origin recognition complex

2018 ◽  
Vol 115 (26) ◽  
pp. E5906-E5915 ◽  
Author(s):  
Franziska Bleichert ◽  
Alexander Leitner ◽  
Ruedi Aebersold ◽  
Michael R. Botchan ◽  
James M. Berger
Keyword(s):  
Dna Binding ◽  
Origin Recognition Complex ◽  
Dna Duplexes ◽  
Dna Interactions ◽  
Minichromosome Maintenance ◽  
Winged Helix ◽  
Replicative Helicase ◽  
Biochemical Assays ◽  
Chemical Cross Linking ◽  
Origin Recognition

In eukaryotes, the heterohexameric origin recognition complex (ORC) coordinates replication onset by facilitating the recruitment and loading of the minichromosome maintenance 2–7 (Mcm2–7) replicative helicase onto DNA to license origins.DrosophilaORC can adopt an autoinhibited configuration that is predicted to prevent Mcm2–7 loading; how the complex is activated and whether other ORC homologs can assume this state are not known. Using chemical cross-linking and mass spectrometry, biochemical assays, and electron microscopy (EM), we show that the autoinhibited state ofDrosophilaORC is populated in solution, and that human ORC can also adopt this form. ATP binding to ORC supports a transition from the autoinhibited state to an active configuration, enabling the nucleotide-dependent association of ORC with both DNA and Cdc6. An unstructured N-terminal region adjacent to the conserved ATPase domain of Orc1 is shown to be required for high-affinity ORC–DNA interactions, but not for activation. ORC optimally binds DNA duplexes longer than the predicted footprint of the ORC ATPases associated with a variety of cellular activities (AAA+) and winged-helix (WH) folds; cryo-EM analysis ofDrosophilaORC bound to DNA and Cdc6 indicates that ORC contacts DNA outside of its central core region, bending the DNA away from its central DNA-binding channel. Our findings indicate that ORC autoinhibition may be common to metazoans and that ORC–Cdc6 remodels origin DNA before Mcm2–7 recruitment and loading.

scholarly journals Dendrite development

2005 ◽  
Vol 170 (4) ◽  
pp. 517-519 ◽  
Author(s):  
Michael D. Ehlers
Keyword(s):  
Dna Replication ◽  
Origin Recognition Complex ◽  
The Core ◽  
Dendrite Development ◽  
Origin Recognition

Neurons extend elaborate dendrites studded with spines. Unexpectedly, this cellular sculpting is regulated by the origin recognition complex—the core machinery for initiating DNA replication.

scholarly journals The origin recognition core complex regulates dendrite and spine development in postmitotic neurons

2005 ◽  
Vol 170 (4) ◽  
pp. 527-535 ◽  
Author(s):  
Zhen Huang ◽  
Keling Zang ◽  
Louis F. Reichardt
Keyword(s):  
Dendritic Spine ◽  
Origin Recognition Complex ◽  
Replication Initiation ◽  
Genome Replication ◽  
Core Complex ◽  
Spine Development ◽  
Branch Formation ◽  
Postmitotic Neurons ◽  
Sirna Knockdown ◽  
Origin Recognition

The origin recognition complex (ORC) ensures exactly one round of genome replication per cell cycle through acting as a molecular switch that precisely controls the assembly, firing, and inactivation of the replication initiation machinery. Recent data indicate that it may also coordinate the processes of mitosis and cytokinesis and ensure the proper distribution of replicated genome to daughter cells. We have found that the ORC core subunits are highly expressed in the nervous system. They are selectively localized to the neuronal somatodendritic compartment and enriched in the membrane fraction. siRNA knockdown of ORC subunits dramatically reduced dendritic branch formation and severely impeded dendritic spine emergence. Expression of ORC ATPase motif mutants enhanced the branching of dendritic arbors. The ORC core complex thus appears to have a novel role in regulating dendrite and dendritic spine development in postmitotic neurons.

scholarly journals Role of the Orc6 Protein in Origin Recognition Complex-Dependent DNA Binding and Replication in Drosophila melanogaster

2007 ◽  
Vol 27 (8) ◽  
pp. 3143-3153 ◽  
Author(s):  
Maxim Balasov ◽  
Richard P. H. Huijbregts ◽  
Igor Chesnokov
Keyword(s):  
Amino Acids ◽  
Dna Replication ◽  
Dna Binding ◽  
Origin Recognition Complex ◽  
Dominant Negative ◽  
The Core ◽  
Culture Cells ◽  
Replication Domain ◽  
Origin Recognition

ABSTRACT The six-subunit origin recognition complex (ORC) is a DNA replication initiator protein in eukaryotes that defines the localization of the origins of replication. We report here that the smallest Drosophila ORC subunit, Orc6, is a DNA binding protein that is necessary for the DNA binding and DNA replication functions of ORC. Orc6 binds DNA fragments containing Drosophila origins of DNA replication and prefers poly(dA) sequences. We have defined the core replication domain of the Orc6 protein which does not include the C-terminal domain. Further analysis of the core replication domain identified amino acids that are important for DNA binding by Orc6. Alterations of these amino acids render reconstituted Drosophila ORC inactive in DNA binding and DNA replication. We show that mutant Orc6 proteins do not associate with chromosomes in vivo and have dominant negative effects in Drosophila tissue culture cells. Our studies provide a molecular analysis for the functional requirement of Orc6 in replicative functions of ORC in Drosophila and suggest that Orc6 may contribute to the sequence preferences of ORC in targeting to the origins.

scholarly journals Author response: The dynamic nature of the human origin recognition complex revealed through five cryoEM structures

2020 ◽  
Author(s):  
Matt J Jaremko ◽  
Kin Fan On ◽  
Dennis R Thomas ◽  
Bruce Stillman ◽  
Leemor Joshua-Tor
Keyword(s):  
Origin Recognition Complex ◽  
Author Response ◽  
Human Origin ◽  
Dynamic Nature ◽  
Origin Recognition

scholarly journals Stabilisation of half MCM ring by Cdt1 during DNA insertion

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Marina Guerrero-Puigdevall ◽  
Narcis Fernandez-Fuentes ◽  
Jordi Frigola
Keyword(s):  
Cell Cycle ◽  
Origin Recognition Complex ◽  
Eukaryotic Cells ◽  
Duplex Dna ◽  
Unknown Mechanism ◽  
Winged Helix ◽  
Replicative Helicase ◽  
Origin Licensing ◽  
Dna Insertion ◽  
Winged Helix Domain

AbstractOrigin licensing ensures precise once per cell cycle replication in eukaryotic cells. The Origin Recognition Complex, Cdc6 and Cdt1 load Mcm2-7 helicase (MCM) into a double hexamer, bound around duplex DNA. The complex formed by ORC-Cdc6 bound to duplex DNA (OC) recruits the MCM-Cdt1 complex into the replication origins. Through the stacking of both complexes, the duplex DNA is inserted inside the helicase by an unknown mechanism. In this paper we show that the DNA insertion comes with a topological problem in the stacking of OC with MCM-Cdt1. Unless an essential, conserved C terminal winged helix domain (C-WHD) of Cdt1 is present, the MCM splits into two halves. The binding of this domain with the essential C-WHD of Mcm6, allows the latching between the MCM-Cdt1 and OC, through a conserved Orc5 AAA-lid interaction. Our work provides new insights into how DNA is inserted into the eukaryotic replicative helicase, through a series of synchronized events.

scholarly journals Structural insight into the assembly and conformational activation of human origin recognition complex

2020 ◽  
Vol 6 (1) ◽  
Author(s):  
Jiaxuan Cheng ◽  
Ningning Li ◽  
Xiaohan Wang ◽  
Jiazhi Hu ◽  
Yuanliang Zhai ◽  
...  
Keyword(s):  
Dna Binding ◽  
Origin Recognition Complex ◽  
Molecular Mechanisms ◽  
Dynamic Structure ◽  
Winged Helix ◽  
Subsequent Activation ◽  
Winged Helix Domain ◽  
Structural Characterizations ◽  
Insight Into ◽  
Origin Recognition

AbstractThe function of the origin recognition complex (ORC) in DNA replication is highly conserved in recognizing and marking the initiation sites. The detailed molecular mechanisms by which human ORC is reconfigured into a state competent for origin association remain largely unknown. Here, we present structural characterizations of human ORC1–5 and ORC2–5 assemblies. ORC2–5 exhibits a tightly autoinhibited conformation with the winged-helix domain of ORC2 completely blocking the central DNA-binding channel. The binding of ORC1 partially relieves the autoinhibitory effect of ORC2–5 through remodeling ORC2-WHD, which makes ORC2-WHD away from the central channel creating a still autoinhibited but more dynamic structure. In particular, the AAA+ domain of ORC1 is highly flexible to sample a variety of conformations from inactive to potentially active states. These results provide insights into the detailed mechanisms regulating the autoinhibition of human ORC and its subsequent activation for DNA binding.

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