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

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.

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

10.1101/2020.08.13.250019 ◽

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.

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

eLife ◽

10.7554/elife.58622 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 2

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.

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

Cell Discovery ◽

10.1038/s41421-020-00232-3 ◽

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

Nature Communications ◽

10.1038/s41467-021-24199-1 ◽

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.

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Structural basis of DNA replication origin recognition by human Orc6 protein binding with DNA

Nucleic Acids Research ◽

10.1093/nar/gkaa751 ◽

2020 ◽

Vol 48 (19) ◽

pp. 11146-11161

Author(s):

Naining Xu ◽

Yingying You ◽

Changdong Liu ◽

Maxim Balasov ◽

Lee Tung Lun ◽

...

Keyword(s):

Dna Replication ◽

Dna Binding ◽

Solution Structure ◽

Origin Recognition Complex ◽

Structural Basis ◽

Dna Replication Origin ◽

Dna Alterations ◽

Replication Initiator ◽

Binding Subunit ◽

Origin Recognition

Abstract The six-subunit origin recognition complex (ORC), a DNA replication initiator, defines the localization of the origins of replication in eukaryotes. The Orc6 subunit is the smallest and the least conserved among ORC subunits. It is required for DNA replication and essential for viability in all species. Orc6 in metazoans carries a structural homology with transcription factor TFIIB and can bind DNA on its own. Here, we report a solution structure of the full-length human Orc6 (HsOrc6) alone and in a complex with DNA. We further showed that human Orc6 is composed of three independent domains: N-terminal, middle and C-terminal (HsOrc6-N, HsOrc6-M and HsOrc6-C). We also identified a distinct DNA-binding domain of human Orc6, named as HsOrc6-DBD. The detailed analysis of the structure revealed novel amino acid clusters important for the interaction with DNA. Alterations of these amino acids abolish DNA-binding ability of Orc6 and result in reduced levels of DNA replication. We propose that Orc6 is a DNA-binding subunit of human/metazoan ORC and may play roles in targeting, positioning and assembling the functional ORC at the origins.

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DrosophilaHeterochromatin Protein 1 (HP1)/Origin Recognition Complex (ORC) Protein Is Associated with HP1 and ORC and Functions in Heterochromatin-induced Silencing

Molecular Biology of the Cell ◽

10.1091/mbc.12.6.1671 ◽

2001 ◽

Vol 12 (6) ◽

pp. 1671-1685 ◽

Cited By ~ 83

Author(s):

Mohammed Momin Shareef ◽

Chadwick King ◽

Mona Damaj ◽

RamaKrishna Badagu ◽

Da Wei Huang ◽

...

Keyword(s):

Dna Binding ◽

Origin Recognition Complex ◽

Polytene Chromosomes ◽

Binding Activity ◽

Hmg Proteins ◽

Mitotic Chromosomes ◽

Origin Firing ◽

Dna Binding Activity ◽

Origin Recognition

Heterochromatin protein 1 (HP1) is a conserved component of the highly compact chromatin of higher eukaryotic centromeres and telomeres. Cytogenetic experiments in Drosophila have shown that HP1 localization into this chromatin is perturbed in mutants for the origin recognition complex (ORC) 2 subunit. ORC has a multisubunit DNA-binding activity that binds origins of DNA replication where it is required for origin firing. The DNA-binding activity of ORC is also used in the recruitment of the Sir1 protein to silence nucleation sites flanking silent copies of the mating-type genes inSaccharomyces cerevisiae. A fraction of HP1 in the maternally loaded cytoplasm of the early Drosophilaembryo is associated with a multiprotein complex containingDrosophila melanogaster ORC subunits. This complex appears to be poised to function in heterochromatin assembly later in embryonic development. Here we report the identification of a novel component of this complex, the HP1/ORC-associated protein. This protein contains similarity to DNA sequence-specific HMG proteins and is shown to bind specific satellite sequences and the telomere-associated sequence in vitro. The protein is shown to have heterochromatic localization in both diploid interphase and mitotic chromosomes and polytene chromosomes. Moreover, the gene encoding HP1/ORC-associated protein was found to display reciprocal dose-dependent variegation modifier phenotypes, similar to those for mutants in HP1 and the ORC 2 subunit.

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Architecture of the yeast origin recognition complex bound to origins of DNA replication.

Molecular and Cellular Biology ◽

10.1128/mcb.17.12.7159 ◽

1997 ◽

Vol 17 (12) ◽

pp. 7159-7168 ◽

Cited By ~ 127

Author(s):

D G Lee ◽

S P Bell

Keyword(s):

Dna Replication ◽

Dna Binding ◽

Origin Recognition Complex ◽

Binding Assays ◽

Chromosomal Replication ◽

Origins Of Replication ◽

Eukaryotic Dna ◽

Level Of Analysis ◽

Origin Recognition

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.

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