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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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Eukaryotic ORC and archaeal Orc WH domains. a) WH domain – DNA interactions in archaeal Orc1-1 (PDB code 2qby chain A)16. The WH domain (grey) uses a helix-turn-helix (HTH, black and white) motif and a β-hairpin wing (cyan) motif to engage DNA (tan surface). b) Disposition of WH domains in ORC. The ORC WH domains (excepting Orc2) form a collar in which the recognition helices (black) are buried and the β-hairpin wings form an exposed portion of the central channel (the Orc1 AAA+ domain is not shown). The HTH motif and β-hairpin wings of ORC subunits are colored as in (a). c) Schematic of the HTH and β-hairpin motifs in ORC.
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Figure 2: Eukaryotic ORC and archaeal Orc WH domains. a) WH domain – DNA interactions in archaeal Orc1-1 (PDB code 2qby chain A)16. The WH domain (grey) uses a helix-turn-helix (HTH, black and white) motif and a β-hairpin wing (cyan) motif to engage DNA (tan surface). b) Disposition of WH domains in ORC. The ORC WH domains (excepting Orc2) form a collar in which the recognition helices (black) are buried and the β-hairpin wings form an exposed portion of the central channel (the Orc1 AAA+ domain is not shown). The HTH motif and β-hairpin wings of ORC subunits are colored as in (a). c) Schematic of the HTH and β-hairpin motifs in ORC.

Mentions: In archaeal Orc homologs, the WH element is responsible for recognizing origin sequences, in which a helix-turn-helix (HTH) motif and a β-hairpin “wing” interact with the adjacent major and minor grooves of double-stranded DNA16,17,20,21 (Fig. 2a). Given the conservation between archaeal Orcs and eukaryotic Orc1-5 proteins, we anticipated that ORC’s WH domains would bind DNA in a similar manner. However, the second α-helix of the HTH in the WH domain (corresponding to the DNA “recognition helix”) is buried against the AAA+ tier in all subunits but Orc2 (Fig. 2b). This arrangement leaves the β-hairpin wings of Orc1, Orc4, Orc5 and Orc3 solvent exposed, which in turn co-localize to form a portion of the interior surface within the central channel in the ORC body (Fig. 2b, c). Given the extensive contacts between the WH and AAA+ tiers, sequestration of the recognition helix seems necessary to maintain ORC integrity. Thus, certain aspects of DNA recognition by the WH domains of ORC likely differ from the approach used by archaeal Orcs.


Crystal structure of the eukaryotic origin recognition complex.

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

Eukaryotic ORC and archaeal Orc WH domains. a) WH domain – DNA interactions in archaeal Orc1-1 (PDB code 2qby chain A)16. The WH domain (grey) uses a helix-turn-helix (HTH, black and white) motif and a β-hairpin wing (cyan) motif to engage DNA (tan surface). b) Disposition of WH domains in ORC. The ORC WH domains (excepting Orc2) form a collar in which the recognition helices (black) are buried and the β-hairpin wings form an exposed portion of the central channel (the Orc1 AAA+ domain is not shown). The HTH motif and β-hairpin wings of ORC subunits are colored as in (a). c) Schematic of the HTH and β-hairpin motifs in ORC.
© Copyright Policy - permissions-link
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4368505&req=5

Figure 2: Eukaryotic ORC and archaeal Orc WH domains. a) WH domain – DNA interactions in archaeal Orc1-1 (PDB code 2qby chain A)16. The WH domain (grey) uses a helix-turn-helix (HTH, black and white) motif and a β-hairpin wing (cyan) motif to engage DNA (tan surface). b) Disposition of WH domains in ORC. The ORC WH domains (excepting Orc2) form a collar in which the recognition helices (black) are buried and the β-hairpin wings form an exposed portion of the central channel (the Orc1 AAA+ domain is not shown). The HTH motif and β-hairpin wings of ORC subunits are colored as in (a). c) Schematic of the HTH and β-hairpin motifs in ORC.
Mentions: In archaeal Orc homologs, the WH element is responsible for recognizing origin sequences, in which a helix-turn-helix (HTH) motif and a β-hairpin “wing” interact with the adjacent major and minor grooves of double-stranded DNA16,17,20,21 (Fig. 2a). Given the conservation between archaeal Orcs and eukaryotic Orc1-5 proteins, we anticipated that ORC’s WH domains would bind DNA in a similar manner. However, the second α-helix of the HTH in the WH domain (corresponding to the DNA “recognition helix”) is buried against the AAA+ tier in all subunits but Orc2 (Fig. 2b). This arrangement leaves the β-hairpin wings of Orc1, Orc4, Orc5 and Orc3 solvent exposed, which in turn co-localize to form a portion of the interior surface within the central channel in the ORC body (Fig. 2b, c). Given the extensive contacts between the WH and AAA+ tiers, sequestration of the recognition helix seems necessary to maintain ORC integrity. Thus, certain aspects of DNA recognition by the WH domains of ORC likely differ from the approach used by archaeal Orcs.

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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