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 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 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 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 Structural basis for DNA recognition by the transcription regulator MetR

2016 ◽  
Vol 72 (6) ◽  
pp. 417-426 ◽  
Author(s):  
Avinash S. Punekar ◽  
Jonathan Porter ◽  
Stephen B. Carr ◽  
Simon E. V. Phillips
Keyword(s):  
Molecular Level ◽  
Dna Recognition ◽  
Data Driven ◽  
Structural Basis ◽  
Methionine Biosynthesis ◽  
Dna Complexes ◽  
Winged Helix ◽  
Target Dna ◽  
Operator Sequence ◽  
Dna Complex

MetR, a LysR-type transcriptional regulator (LTTR), has been extensively studied owing to its role in the control of methionine biosynthesis in proteobacteria. A MetR homodimer binds to a 24-base-pair operator region of themetgenes and specifically recognizes the interrupted palindromic sequence 5′-TGAA-N5-TTCA-3′. Mechanistic details underlying the interaction of MetR with its target DNA at the molecular level remain unknown. In this work, the crystal structure of the DNA-binding domain (DBD) of MetR was determined at 2.16 Å resolution. MetR-DBD adopts a winged-helix–turn–helix (wHTH) motif and shares significant fold similarity with the DBD of the LTTR protein BenM. Furthermore, a data-driven macromolecular-docking strategy was used to model the structure of MetR-DBD bound to DNA, which revealed that a bent conformation of DNA is required for the recognition helix α3 and the wing loop of the wHTH motif to interact with the major and minor grooves, respectively. Comparison of the MetR-DBD–DNA complex with the crystal structures of other LTTR-DBD–DNA complexes revealed residues that may confer operator-sequence binding specificity for MetR. Taken together, the results show that MetR-DBD uses a combination of direct base-specific interactions and indirect shape recognition of the promoter to regulate the transcription ofmetgenes.

scholarly journals The Purine Repressor of Bacillus subtilis: a Novel Combination of Domains Adapted for Transcription Regulation

2003 ◽  
Vol 185 (14) ◽  
pp. 4087-4098 ◽  
Author(s):  
Sangita C. Sinha ◽  
Joseph Krahn ◽  
Byung Sik Shin ◽  
Diana R. Tomchick ◽  
Howard Zalkin ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Dna Binding ◽  
Dna Sequences ◽  
Sequence Motif ◽  
Charged Surface ◽  
Winged Helix ◽  
Gram Positive Bacteria ◽  
Terminal Domain ◽  
Winged Helix Domain ◽  
Positively Charged

ABSTRACT The purine repressor from Bacillus subtilis, PurR, represses transcription from a number of genes with functions in the synthesis, transport, and metabolism of purines. The 2.2-Å crystal structure of PurR reveals a two-domain protein organized as a dimer. The larger C-terminal domain belongs to the PRT structural family, in accord with a sequence motif for binding the inducer phosphoribosylpyrophosphate (PRPP). The PRT domain is fused to a smaller N-terminal domain that belongs to the winged-helix family of DNA binding proteins. A positively charged surface on the winged-helix domain likely binds specific DNA sequences in the recognition site. A second positively charged surface surrounds the PRPP site at the opposite end of the PurR dimer. Conserved amino acids in the sequences of PurR homologs in 21 gram-positive bacteria cluster on the proposed recognition surface of the winged-helix domain and around the PRPP binding site at the opposite end of the molecule, supporting a common function of DNA and PRPP binding for all of the proteins. The structure supports a binding mechanism in which extended regions of DNA interact with extensive protein surface. Unlike most PRT proteins, which are phosphoribosyltransferases (PRTases), PurR lacks catalytic activity. This is explained by a tyrosine side chain that blocks the site for a nucleophile cosubstrate in PRTases. Thus, B. subtilis has adapted an enzyme fold to serve as an effector-binding domain and has used it in a novel combination with the DNA-binding winged-helix domain as a repressor of purine genes.

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.

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 A Genomic Distance Based on MUM Indicates Discontinuity between Most Bacterial Species and Genera

2008 ◽  
Vol 191 (1) ◽  
pp. 91-99 ◽  
Author(s):  
Marc Deloger ◽  
Meriem El Karoui ◽  
Marie-Agnès Petit
Keyword(s):  
Dna Sequences ◽  
Dna Content ◽  
Core Genome ◽  
Biological Diversity ◽  
Bacterial Species ◽  
Genomic Distance ◽  
The Core ◽  
Intraspecies Diversity ◽  
Genome Level ◽  
Definition Of

ABSTRACT The fundamental unit of biological diversity is the species. However, a remarkable extent of intraspecies diversity in bacteria was discovered by genome sequencing, and it reveals the need to develop clear criteria to group strains within a species. Two main types of analyses used to quantify intraspecies variation at the genome level are the average nucleotide identity (ANI), which detects the DNA conservation of the core genome, and the DNA content, which calculates the proportion of DNA shared by two genomes. Both estimates are based on BLAST alignments for the definition of DNA sequences common to the genome pair. Interestingly, however, results using these methods on intraspecies pairs are not well correlated. This prompted us to develop a genomic-distance index taking into account both criteria of diversity, which are based on DNA maximal unique matches (MUM) shared by two genomes. The values, called MUMi, for MUM index, correlate better with the ANI than with the DNA content. Moreover, the MUMi groups strains in a way that is congruent with routinely used multilocus sequence-typing trees, as well as with ANI-based trees. We used the MUMi to determine the relatedness of all available genome pairs at the species and genus levels. Our analysis reveals a certain consistency in the current notion of bacterial species, in that the bulk of intraspecies and intragenus values are clearly separable. It also confirms that some species are much more diverse than most. As the MUMi is fast to calculate, it offers the possibility of measuring genome distances on the whole database of available genomes.

Sign in / Sign up

Export Citation Format

Share Document