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. 2018 Jun 26;115(26):E5906-E5915.
doi: 10.1073/pnas.1806315115. Epub 2018 Jun 13.

Conformational Control and DNA-binding Mechanism of the Metazoan Origin Recognition Complex

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Free PMC article

Conformational Control and DNA-binding Mechanism of the Metazoan Origin Recognition Complex

Franziska Bleichert et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

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. Drosophila ORC 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 of Drosophila ORC 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 of Drosophila ORC 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.

Keywords: AAA+ ATPase; DNA replication; helicase loading; initiators; origin recognition complex.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
The autoinhibited ORC state is a conserved characteristic of ORC and can be detected in solution. (A and B) Cross-linking mass spectrometry of the DmORC core complex reveals specific cross-links expected for the autoinhibited (red dashed lines) and active (cyan dashed line) conformational states. In A, cross-links reporting on the conformational state of ORC are mapped onto the DmORC crystal structure (Left, autoinhibited) and on a model of an “activated” DmORC complex (Right; Materials and Methods). The Orc2 WH domain is omitted for clarity. In B, all DSS-induced cross-links are mapped onto the domain architecture of ORC subunits, with interprotein cross-links depicted as straight blue lines and intraprotein cross-links shown in magenta. The positions of the first and last amino acids of each subunit in the ORC core complex are indicated, with ordered domains and disordered linker regions shown either in color or in white, respectively. Note that one of the cross-links supporting the autoinhibited conformation was detected twice on slightly different peptides due to alternative protease cleavage. (C) Negative-stain EM analysis of DmORC and human ORC1–5 indicates that both complexes can adopt the autoinhibited conformation in the presence of the ATP analog ATPγS. Class averages reflecting the autoinhibited state are shown in two different views, with the characteristic Orc1 density that defines the autoinhibited state highlighted by an orange arrowhead. Top and side views of the crystal structure of Drosophila ORC (6) in the autoinhibited conformation (Right) and an active DmORC model (Left) are displayed for comparison. Subunits are colored as in A.
Fig. 2.
Fig. 2.
ATP and, to a lesser extent, ATPγS, stabilize Drosophila ORC in the active conformation and allow ORC association with both DNA and Cdc6. (A) Top and side views of the DmORC crystal structure (6) (Top Left) and of a model of activated DmORC (Top Right) are shown. Low-pass-filtered 2D projections of both ORC models in top and side views, as well as corresponding class averages of negatively stained DmORC in the presence of ATPγS or ATP, are depicted below. Arrowheads point to the Orc1 AAA+ density that repositions between ATP and ATPγS states (differences in top and side views are indicated in yellow and orange, respectively). Note that no class average depicting the side view of the active state was observed with ATPγS-DmORC. (B) Duplex DNA binding by DmORC was assayed by fluorescence anisotropy (FA) in the absence or presence of different nucleotides at 1 mM concentration. High (low-nanomolar) affinity binding is observed in the presence of ATP, but not ADP or ATPγS. Kds were calculated for the ATP, ADP–BeF3, and ATPγS conditions, but the lack of a plateau in the binding curves obtained with ADP or without nucleotide prevented accurate Kd determination for these conditions. (C) Pull-down assays using MBP-tagged DmCdc6 as bait demonstrate that ATP and, to a lesser extent, ATPγS, can promote the coassociation of ORC with Cdc6. All reactions were performed in the presence of DNA. Input and eluted proteins were separated by SDS/PAGE and visualized by silver staining.
Fig. 3.
Fig. 3.
The N-terminal region of Orc1 is required for high-affinity, ATP-dependent DNA binding by ORC. (A) Schematic of ORC domain architecture with the ORC core complex lacking the N-terminal regions of Orc1 [including the bromoadjacent homology (BAH) domain], Orc2, Orc3, and Orc6 (including the TFIIB-like domain) bordered in gray. CTD, C-terminal domain; WH, winged-helix domain. ATP-dependent DNA binding by full-length DmORC (WT), the DmORC core (DmORCOrc1ΔN, Orc2ΔN, Orc3ΔN, Orc6ΔN; abbreviated as 1ΔN, 2ΔN, 3ΔN, 6ΔN), or DmORC lacking different combinations of N-terminal regions for subunits Orc1 (1ΔN), Orc2 (2ΔN), Orc3 (3ΔN), and Orc6 (6ΔN) was analyzed by fluorescence anisotropy (FA) (B) and pull-down assays (C). Kds and SEs of the parameter fits for binding curves determined by FA are listed in B. ND, Kds could not be determined because binding curves did not saturate. For the pull-downs in C, a biotinylated DNA duplex was used as bait. Copurifying proteins were eluted by UV cleavage, and input and eluted proteins were analyzed by SDS/PAGE followed by silver staining. (D) Two-dimensional EM analysis of negatively stained DmORC lacking the N-terminal region of Orc1 indicates that the removal of Orc1’s N terminus does not prevent ORC from adopting an active conformation in the presence of ATP. Representative top (T), intermediate (I), and side (S) view class averages of DmORCOrc1ΔN in both the active and autoinhibited states are shown. Arrowheads highlight the repositioning of the Orc1 AAA+ density between autoinhibited and active states (differences in top and intermediate/side views are indicated in yellow and orange, respectively). (E) Pull-down assays using MBP-tagged DmCdc6 as bait were used to assess the ability of full-length DmORC, the DmORC core complex [ORCOrc1ΔN, Orc2ΔN, Orc3ΔN, Orc6ΔN (abbreviated as 1ΔN, 2ΔN, 3ΔN, 6ΔN)], and DmORC lacking different N-terminal regions [ORCOrc1ΔN (abbreviated as 1ΔN), ORCOrc6ΔN (abbreviated as 6ΔN), and ORCOrc2ΔN, Orc3ΔN, Orc6ΔN (abbreviated as 2ΔN, 3ΔN, 6ΔN)] to coassociate with Cdc6 in the presence of DNA. Input and eluted proteins were analyzed by SDS/PAGE followed by silver staining. Note that the Orc6 C-terminal peptide (Orc6ΔN) is not resolved and visible on the gels shown in D and E due to its small size.
Fig. 4.
Fig. 4.
A basic patch in the N-terminal region of Orc1 stabilizes ATP-dependent ORC-DNA contacts. (A) ATP-dependent DNA binding of ORC (full-length or containing N-terminally truncated Orc1) to a 40-bp DNA duplex was measured by fluorescence anisotropy (FA). Kds and SEs of the parameter fits are listed for each mutant complex except for ORCOrc1ΔN528 and ORCOrc1ΔN532, for which the lack of a plateau prevented accurate Kd determination. ND, not determined. (B) Schematic of the Orc1 domain architecture depicting the relative positions of conserved bromoadjacent homology (BAH), AAA+ ATPase, and WH domains. Truncations used in A are indicated, as is the region that stimulates ATP-dependent DNA binding (dotted box). A sequence LOGO of this region (corresponding to amino acids 490–530 in DmOrc1), generated using an alignment of metazoan Orc1 protein sequences, reveals several highly conserved basic amino acid residues, including R492, K523, and R528 in DmOrc1. Asterisks denote basic residues that are either missing in several metazoan Orc1 sequences or are not conserved in DmOrc1. (C) Changing conserved basic Orc1 amino acid residues to glutamate decreases the ATP-dependent DNA-binding activity of metazoan ORC. DmORC containing the Orc1R492E/K523E/R528E triple mutant (1RKR-EEE) was purified, and its DNA-binding activity was tested in the presence of ATP by FA as in A.
Fig. 5.
Fig. 5.
ATP-dependent DNA binding by ORC–Cdc6 leads to DNA bending. (A) Docking of a DNA duplex into DmORC’s central channel (based on ref. 6) shows that it can only accommodate 20–25 bp of a DNA duplex. (B) DNA length requirements for ATP-dependent DNA binding by full-length DmORC were assessed by fluorescence anisotropy (FA) using fluorescein-labeled DNA duplexes of indicated lengths in the presence of ATP. Kds and SEs of the parameter fits are listed for DNA duplexes that lead to saturation of DNA binding. ND, Kds could not be determined because binding curves did not saturate. (C) Cryo-EM analysis of DmORC in the presence of DNA and DmCdc6 shows that DNA is bound in ORC’s central channel and is bent toward the domain-swapped Orc2 AAA+/Orc3 WH and Orc3 AAA+/Orc5 WH domains upon exit from one side of the complex. Representative 2D class averages are shown. The locations of ORC subunits and Cdc6 are revealed by superpositioning of a structural model of activated DmORC and highlighted by arrowheads.
Fig. 6.
Fig. 6.
Model for ORC activation, ATP-dependent DNA binding, and Mcm2–7 loading. ATP binding to the Orc1/4 ATPase site and other as yet unidentified factors allows ORC to more readily sample an active configuration that enables the ATP-dependent binding of DNA to the Orc1 basic patch and to elements in the AAA+/WH domain channel, as well as Cdc6 association. DNA and Cdc6 binding is accompanied by bending of the DNA toward the Orc2 AAA+/Orc3 WH and Orc3 AAA+/Orc5 WH units (the WH elements domain swap with the AAA+ modules of adjacent subunits), which allows an Mcm2–7 hexamer to dock onto the ORC–Cdc6 ring in a manner that aligns the DNA duplex with the Mcm2/5 gate. Conformational changes in ORC likely alleviate DNA-ORC contacts required for bending, allowing a straightened DNA segment to engage the Mcm2–7 pore.

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