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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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Structure of Drosophila ORC. a) Domain organization of ORC subunits. Dashed lines demarcate the ORC core used for crystallization (TFIIB and CTD – transcription factor II-like and C-terminal domains in Orc6; BAH – bromo-adjacent homology domain in Orc1). b) Crystal structure of ORC. Domains of individual subunits are colored as in (a). c) Side view of ORC (surface) highlighting the two-tiered, domain-swapped organization of the ORC body (Orc1 AAA+ and Orc2 WH domains, as well as N-terminal Orc2 residues (built as polyalanine) are not shown). Exploded view (cartoon) showing the packing of WH domains against adjacent subunits’ AAA+ regions.
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Figure 1: Structure of Drosophila ORC. a) Domain organization of ORC subunits. Dashed lines demarcate the ORC core used for crystallization (TFIIB and CTD – transcription factor II-like and C-terminal domains in Orc6; BAH – bromo-adjacent homology domain in Orc1). b) Crystal structure of ORC. Domains of individual subunits are colored as in (a). c) Side view of ORC (surface) highlighting the two-tiered, domain-swapped organization of the ORC body (Orc1 AAA+ and Orc2 WH domains, as well as N-terminal Orc2 residues (built as polyalanine) are not shown). Exploded view (cartoon) showing the packing of WH domains against adjacent subunits’ AAA+ regions.

Mentions: Sequence analyses had indicated that the Orc1-5 subunits would share a domain architecture similar to that of archaeal Orc proteins, with AAA+-type ATPase folds fused to at least one C-terminal winged-helix (WH) DNA-binding domain (Fig. 1a)7,8. For its part, the Orc6 C-terminus has been reported to bind to ORC1-5 through a domain insertion in Orc3, leaving its TFIIB-like domain conformationally independent of the ORC core (Fig. 1a)14. For crystallizing Drosophila ORC, we designed a “trimmed” construct lacking the flexible N-terminal extensions of Orc1, Orc2 and Orc314, and the Orc6 TFIIB region (Fig. 1a). Neither modification interfered with ORC assembly, nor did they affect the overall architecture of ORC (Extended Data Fig. 1a–c). This ORC core (referred to as ORC hereafter) crystalized in space group I222 with one Orc1-6 heterohexamer per asymmetric unit. The structure was phased by single-wavelength anomalous dispersion and refined to 3.5 Å with an Rwork/Rfree of 0.22/0.26 (Extended Data Fig. 2a–c and Extended Data Table 1).


Crystal structure of the eukaryotic origin recognition complex.

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

Structure of Drosophila ORC. a) Domain organization of ORC subunits. Dashed lines demarcate the ORC core used for crystallization (TFIIB and CTD – transcription factor II-like and C-terminal domains in Orc6; BAH – bromo-adjacent homology domain in Orc1). b) Crystal structure of ORC. Domains of individual subunits are colored as in (a). c) Side view of ORC (surface) highlighting the two-tiered, domain-swapped organization of the ORC body (Orc1 AAA+ and Orc2 WH domains, as well as N-terminal Orc2 residues (built as polyalanine) are not shown). Exploded view (cartoon) showing the packing of WH domains against adjacent subunits’ AAA+ regions.
© Copyright Policy - permissions-link
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4368505&req=5

Figure 1: Structure of Drosophila ORC. a) Domain organization of ORC subunits. Dashed lines demarcate the ORC core used for crystallization (TFIIB and CTD – transcription factor II-like and C-terminal domains in Orc6; BAH – bromo-adjacent homology domain in Orc1). b) Crystal structure of ORC. Domains of individual subunits are colored as in (a). c) Side view of ORC (surface) highlighting the two-tiered, domain-swapped organization of the ORC body (Orc1 AAA+ and Orc2 WH domains, as well as N-terminal Orc2 residues (built as polyalanine) are not shown). Exploded view (cartoon) showing the packing of WH domains against adjacent subunits’ AAA+ regions.
Mentions: Sequence analyses had indicated that the Orc1-5 subunits would share a domain architecture similar to that of archaeal Orc proteins, with AAA+-type ATPase folds fused to at least one C-terminal winged-helix (WH) DNA-binding domain (Fig. 1a)7,8. For its part, the Orc6 C-terminus has been reported to bind to ORC1-5 through a domain insertion in Orc3, leaving its TFIIB-like domain conformationally independent of the ORC core (Fig. 1a)14. For crystallizing Drosophila ORC, we designed a “trimmed” construct lacking the flexible N-terminal extensions of Orc1, Orc2 and Orc314, and the Orc6 TFIIB region (Fig. 1a). Neither modification interfered with ORC assembly, nor did they affect the overall architecture of ORC (Extended Data Fig. 1a–c). This ORC core (referred to as ORC hereafter) crystalized in space group I222 with one Orc1-6 heterohexamer per asymmetric unit. The structure was phased by single-wavelength anomalous dispersion and refined to 3.5 Å with an Rwork/Rfree of 0.22/0.26 (Extended Data Fig. 2a–c and Extended Data Table 1).

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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