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Crystal structure of the eukaryotic origin recognition complex.

Bleichert F, Botchan MR, Berger JM - Nature (2015)

Bottom Line: These include highly interdigitated domain-swapping interactions between the winged-helix folds and AAA+ modules of neighbouring protomers, and a quasi-spiral arrangement of DNA binding elements that circumnavigate an approximately 20 Å wide channel in the centre of the complex.Comparative analyses indicate that ORC encircles DNA, using its winged-helix domain face to engage the mini-chromosome maintenance 2-7 (MCM2-7) complex during replicative helicase loading; however, an observed out-of-plane rotation of more than 90° for the Orc1 AAA+ domain disrupts interactions with catalytic amino acids in Orc4, narrowing and sealing off entry into the central channel.Prima facie, our data indicate that Drosophila ORC can switch between active and autoinhibited conformations, suggesting a novel means for cell cycle and/or developmental control of ORC functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biophysics and Biophysical Chemistry, Johns Hopkins School of Medicine, Baltimore, Maryland 21205, USA.

ABSTRACT
Initiation of cellular DNA replication is tightly controlled to sustain genomic integrity. In eukaryotes, the heterohexameric origin recognition complex (ORC) is essential for coordinating replication onset. Here we describe the crystal structure of Drosophila ORC at 3.5 Å resolution, showing that the 270 kilodalton initiator core complex comprises a two-layered notched ring in which a collar of winged-helix domains from the Orc1-5 subunits sits atop a layer of AAA+ (ATPases associated with a variety of cellular activities) folds. Although canonical inter-AAA+ domain interactions exist between four of the six ORC subunits, unanticipated features are also evident. These include highly interdigitated domain-swapping interactions between the winged-helix folds and AAA+ modules of neighbouring protomers, and a quasi-spiral arrangement of DNA binding elements that circumnavigate an approximately 20 Å wide channel in the centre of the complex. Comparative analyses indicate that ORC encircles DNA, using its winged-helix domain face to engage the mini-chromosome maintenance 2-7 (MCM2-7) complex during replicative helicase loading; however, an observed out-of-plane rotation of more than 90° for the Orc1 AAA+ domain disrupts interactions with catalytic amino acids in Orc4, narrowing and sealing off entry into the central channel. Prima facie, our data indicate that Drosophila ORC can switch between active and autoinhibited conformations, suggesting a novel means for cell cycle and/or developmental control of ORC functions.

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Docking of the ORC structure into the cryo-EM structure of an S. cerevisiae replication initiation intermediate indicates that ORC recruits the MCM2-7 complex by binding to the ORC winged-helix (WH) domains. A prior model for ORC•MCM2-7 engagement24, proposed from an ORC•Cdc6•Cdt1•MCM2-7 cryo-EM structure generated in the presence of DNA (shown in (a), EMD-562524), used the crystal structure of replication factor C (RFC) bound to the sliding clamp PCNA (shown in (b), PDB code 1SXJ31) to suggest that ORC’s AAA+ domains engage the MCM2-7•Cdt1 complex. However, using the handedness of the EM volume as reported24, this organization of ORC subunits leads to an inverted ATPase site assembly, requiring that the Orc4 arginine finger (which is known to stimulate Orc1 ATP hydrolysis33) points toward the Orc5 nucleotide-binding site rather than the appropriate Orc1 active site. Schematics for the ATP site assemblies of ORC and RFC derived from these structures are shown in the lower panels in (a) and (b). The location of the WH domain collar of ORC and the C-terminal collar of RFC is indicated by a gray circle (WA – Walker A, WB – Walker B, RF – arginine finger). c) Docking of the ORC crystal structure (with Orc1 in its remodeled or “activated” conformation) into the cryo-EM map shown in panel (a) reveals that the WH domains of ORC face an MCM2-7 complex. This switched polarity of WH domains and AAA+ domains in the EM map corrects the ATPase site assembly and is schematized in the right panel.
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Figure 15: Docking of the ORC structure into the cryo-EM structure of an S. cerevisiae replication initiation intermediate indicates that ORC recruits the MCM2-7 complex by binding to the ORC winged-helix (WH) domains. A prior model for ORC•MCM2-7 engagement24, proposed from an ORC•Cdc6•Cdt1•MCM2-7 cryo-EM structure generated in the presence of DNA (shown in (a), EMD-562524), used the crystal structure of replication factor C (RFC) bound to the sliding clamp PCNA (shown in (b), PDB code 1SXJ31) to suggest that ORC’s AAA+ domains engage the MCM2-7•Cdt1 complex. However, using the handedness of the EM volume as reported24, this organization of ORC subunits leads to an inverted ATPase site assembly, requiring that the Orc4 arginine finger (which is known to stimulate Orc1 ATP hydrolysis33) points toward the Orc5 nucleotide-binding site rather than the appropriate Orc1 active site. Schematics for the ATP site assemblies of ORC and RFC derived from these structures are shown in the lower panels in (a) and (b). The location of the WH domain collar of ORC and the C-terminal collar of RFC is indicated by a gray circle (WA – Walker A, WB – Walker B, RF – arginine finger). c) Docking of the ORC crystal structure (with Orc1 in its remodeled or “activated” conformation) into the cryo-EM map shown in panel (a) reveals that the WH domains of ORC face an MCM2-7 complex. This switched polarity of WH domains and AAA+ domains in the EM map corrects the ATPase site assembly and is schematized in the right panel.

Mentions: Collectively, the structure and analysis presented here provides a framework for understanding how Drosophila ORC interfaces with its partner proteins and DNA during the initial stages of MCM2-7 loading (Fig. 6c). In this scheme, ORC would start off in an ATP-bound but autoinhibited form – either dissociated from chromatin or in a chromatin-bound state via secondary binding sites/partners – that is restricted in its ability to either bind DNA in its central channel or bind Cdc6 to its ring. Conversion of this state into an activated configuration would involve the en bloc movement of the Orc1 ATPase domain (Supplementary Video 1), allowing Orc1 to engage the arginine finger of Orc4, and unlatching the Orc2 WH domain to open a gap in the Orc1-5 ring. Once open, DNA would bind to the ISM and β-hairpin elements in the central ORC channel, after which Cdc6 would dock into the Orc1/Orc2 gap, trapping DNA within the center of the complex. One prediction of this model is that Cdc6 should bind to ORC using its ATPase center to engage an arginine finger in Orc1 (Drosophila residue Arg734). After formation of a ternary ORC•DNA•Cdc6 complex, the WH domains of Cdc6 and Orc2 would be expected to engage the AAA+ folds of Orc1 and Cdc6, respectively, creating a circuit of WH domains (and their associated β-hairpin elements) that help lock the complex into place. Interestingly, our structural findings and analyses reinterpret a recent 3D EM reconstruction concerning the disposition of ORC and MCM2-7 in a helicase-loading intermediate complex24: rather than using its AAA+ domains to bind MCM2-7, which requires an inverted order of ATPase site-arginine finger interactions around the ORC ring (Extended Data Fig. 9a, b), ORC likely uses its WH domain collar instead (Extended Data Fig. 9c).


Crystal structure of the eukaryotic origin recognition complex.

Bleichert F, Botchan MR, Berger JM - Nature (2015)

Docking of the ORC structure into the cryo-EM structure of an S. cerevisiae replication initiation intermediate indicates that ORC recruits the MCM2-7 complex by binding to the ORC winged-helix (WH) domains. A prior model for ORC•MCM2-7 engagement24, proposed from an ORC•Cdc6•Cdt1•MCM2-7 cryo-EM structure generated in the presence of DNA (shown in (a), EMD-562524), used the crystal structure of replication factor C (RFC) bound to the sliding clamp PCNA (shown in (b), PDB code 1SXJ31) to suggest that ORC’s AAA+ domains engage the MCM2-7•Cdt1 complex. However, using the handedness of the EM volume as reported24, this organization of ORC subunits leads to an inverted ATPase site assembly, requiring that the Orc4 arginine finger (which is known to stimulate Orc1 ATP hydrolysis33) points toward the Orc5 nucleotide-binding site rather than the appropriate Orc1 active site. Schematics for the ATP site assemblies of ORC and RFC derived from these structures are shown in the lower panels in (a) and (b). The location of the WH domain collar of ORC and the C-terminal collar of RFC is indicated by a gray circle (WA – Walker A, WB – Walker B, RF – arginine finger). c) Docking of the ORC crystal structure (with Orc1 in its remodeled or “activated” conformation) into the cryo-EM map shown in panel (a) reveals that the WH domains of ORC face an MCM2-7 complex. This switched polarity of WH domains and AAA+ domains in the EM map corrects the ATPase site assembly and is schematized in the right panel.
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Related In: Results  -  Collection

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Figure 15: Docking of the ORC structure into the cryo-EM structure of an S. cerevisiae replication initiation intermediate indicates that ORC recruits the MCM2-7 complex by binding to the ORC winged-helix (WH) domains. A prior model for ORC•MCM2-7 engagement24, proposed from an ORC•Cdc6•Cdt1•MCM2-7 cryo-EM structure generated in the presence of DNA (shown in (a), EMD-562524), used the crystal structure of replication factor C (RFC) bound to the sliding clamp PCNA (shown in (b), PDB code 1SXJ31) to suggest that ORC’s AAA+ domains engage the MCM2-7•Cdt1 complex. However, using the handedness of the EM volume as reported24, this organization of ORC subunits leads to an inverted ATPase site assembly, requiring that the Orc4 arginine finger (which is known to stimulate Orc1 ATP hydrolysis33) points toward the Orc5 nucleotide-binding site rather than the appropriate Orc1 active site. Schematics for the ATP site assemblies of ORC and RFC derived from these structures are shown in the lower panels in (a) and (b). The location of the WH domain collar of ORC and the C-terminal collar of RFC is indicated by a gray circle (WA – Walker A, WB – Walker B, RF – arginine finger). c) Docking of the ORC crystal structure (with Orc1 in its remodeled or “activated” conformation) into the cryo-EM map shown in panel (a) reveals that the WH domains of ORC face an MCM2-7 complex. This switched polarity of WH domains and AAA+ domains in the EM map corrects the ATPase site assembly and is schematized in the right panel.
Mentions: Collectively, the structure and analysis presented here provides a framework for understanding how Drosophila ORC interfaces with its partner proteins and DNA during the initial stages of MCM2-7 loading (Fig. 6c). In this scheme, ORC would start off in an ATP-bound but autoinhibited form – either dissociated from chromatin or in a chromatin-bound state via secondary binding sites/partners – that is restricted in its ability to either bind DNA in its central channel or bind Cdc6 to its ring. Conversion of this state into an activated configuration would involve the en bloc movement of the Orc1 ATPase domain (Supplementary Video 1), allowing Orc1 to engage the arginine finger of Orc4, and unlatching the Orc2 WH domain to open a gap in the Orc1-5 ring. Once open, DNA would bind to the ISM and β-hairpin elements in the central ORC channel, after which Cdc6 would dock into the Orc1/Orc2 gap, trapping DNA within the center of the complex. One prediction of this model is that Cdc6 should bind to ORC using its ATPase center to engage an arginine finger in Orc1 (Drosophila residue Arg734). After formation of a ternary ORC•DNA•Cdc6 complex, the WH domains of Cdc6 and Orc2 would be expected to engage the AAA+ folds of Orc1 and Cdc6, respectively, creating a circuit of WH domains (and their associated β-hairpin elements) that help lock the complex into place. Interestingly, our structural findings and analyses reinterpret a recent 3D EM reconstruction concerning the disposition of ORC and MCM2-7 in a helicase-loading intermediate complex24: rather than using its AAA+ domains to bind MCM2-7, which requires an inverted order of ATPase site-arginine finger interactions around the ORC ring (Extended Data Fig. 9a, b), ORC likely uses its WH domain collar instead (Extended Data Fig. 9c).

Bottom Line: These include highly interdigitated domain-swapping interactions between the winged-helix folds and AAA+ modules of neighbouring protomers, and a quasi-spiral arrangement of DNA binding elements that circumnavigate an approximately 20 Å wide channel in the centre of the complex.Comparative analyses indicate that ORC encircles DNA, using its winged-helix domain face to engage the mini-chromosome maintenance 2-7 (MCM2-7) complex during replicative helicase loading; however, an observed out-of-plane rotation of more than 90° for the Orc1 AAA+ domain disrupts interactions with catalytic amino acids in Orc4, narrowing and sealing off entry into the central channel.Prima facie, our data indicate that Drosophila ORC can switch between active and autoinhibited conformations, suggesting a novel means for cell cycle and/or developmental control of ORC functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biophysics and Biophysical Chemistry, Johns Hopkins School of Medicine, Baltimore, Maryland 21205, USA.

ABSTRACT
Initiation of cellular DNA replication is tightly controlled to sustain genomic integrity. In eukaryotes, the heterohexameric origin recognition complex (ORC) is essential for coordinating replication onset. Here we describe the crystal structure of Drosophila ORC at 3.5 Å resolution, showing that the 270 kilodalton initiator core complex comprises a two-layered notched ring in which a collar of winged-helix domains from the Orc1-5 subunits sits atop a layer of AAA+ (ATPases associated with a variety of cellular activities) folds. Although canonical inter-AAA+ domain interactions exist between four of the six ORC subunits, unanticipated features are also evident. These include highly interdigitated domain-swapping interactions between the winged-helix folds and AAA+ modules of neighbouring protomers, and a quasi-spiral arrangement of DNA binding elements that circumnavigate an approximately 20 Å wide channel in the centre of the complex. Comparative analyses indicate that ORC encircles DNA, using its winged-helix domain face to engage the mini-chromosome maintenance 2-7 (MCM2-7) complex during replicative helicase loading; however, an observed out-of-plane rotation of more than 90° for the Orc1 AAA+ domain disrupts interactions with catalytic amino acids in Orc4, narrowing and sealing off entry into the central channel. Prima facie, our data indicate that Drosophila ORC can switch between active and autoinhibited conformations, suggesting a novel means for cell cycle and/or developmental control of ORC functions.

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