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Nucleotide-Dependent Conformational Changes in the Dnaa-Like Core of the Origin Recognition Complex

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Nucleotide-Dependent Conformational Changes in the Dnaa-Like Core of the Origin Recognition Complex ARTICLES Nucleotide-dependent conformational changes in the DnaA-like core of the origin recognition complex Megan G Clarey1, Jan P Erzberger1, Patricia Grob1, Andres E Leschziner2, James M Berger1, Eva Nogales1–3 & Michael Botchan1 Structural details of initiator proteins for DNA replication have provided clues to the molecular events in this process. EM reconstructions of the Drosophila melanogaster origin recognition complex (ORC) reveal nucleotide-dependent conformational changes in the core of the complex. All five AAA+ domains in ORC contain a conserved structural element that, in DnaA, http://www.nature.com/nsmb promotes formation of a right-handed helix, indicating that helical AAA+ substructures may be a feature of all initiators. A DnaA helical pentamer can be docked into ORC, and the location of Orc5 uniquely positions this core. The results suggest that ATP- dependent conformational changes observed in ORC derive from reorientation of the AAA+ domains. By analogy to the DNA- wrapping activity of DnaA, we posit that ORC together with Cdc6 prepares origin DNA for helicase loading through mechanisms related to the established pathway of prokaryotes. ORC is a conserved, multiprotein complex used to initiate DNA DNA replication, melting of the template provides a portal for replication in all eukaryotes characterized to date1–3. Binding of this loading the helicase. The precise molecular architecture of the eukar- six-subunit assembly to DNA origins marks the first step in DNA yotic helicase has not been resolved completely, but recent data replication and is followed by ORC-directed recruitment of the pre- show that a complex containing the six MCM proteins (MCM2– Nature Publishing Group Group Nature Publishing 6 replication complex (pre-RC) and eventual establishment of bidirect- MCM7), Cdc45 and the four GINS proteins performs this 3 16–18 200 ional DNA replication forks . unwinding activity . In this complex, the ATP-dependent © Despite differences in the recruitment and regulation of replisomal functions of the helicase are provided by the MCM proteins. These factors, numerous structural and functional similarities indicate that MCM proteins associate with the template before helicase there are important mechanistic parallels between prokaryotic and activation and before the acquisition of Cdc45 and GINS, and the eukaryotic DNA replication initiation3–6. For example, all initiator tight binding of the MCMs to the pre-RC requires, at minimum, proteins function as either homomeric or heteromeric complexes of the ORC and Cdc6 proteins19,20. To date, ORC and Cdc6 have AAA+ subunits that are activated through ATP binding7,8.Flankingthe not been found to melt or otherwise deform DNA in preparation AAA+ core, initiators have C-terminal DNA-targeting modules that for MCM loading, as would be anticipated by a strict analogy to mediate origin interactions. In bacteria, DNA targeting precedes ATP- DnaA. Consequently, a widely held speculation has been that the dependent oligomerization of the initiator molecule, DnaA, whereas in AAA+ ensemble of ORC plus Cdc6 functions as the ‘loader’ of a eukaryotes, ATP modulates origin interactions in the context of the ring-like helicase, much as the five-subunit AAA+ replication preformed ORC assembly9–12. Notably, recent studies of human ORC factor C (RFC) brings the PCNA sliding clamp to encircle the have shown that, in addition to stimulating DNA-binding activity, ATP DNA-primer template at the replication fork. The details of binding also has an essential structural role in the formation and how ORC and Cdc6 work to stably engage the MCM proteins stability of the complex12. This finding parallels the assembly mechan- have not yet been revealed. We wished to elucidate the structure ism of DnaA8,13.Additionally,bothDnaAandORChaveanincreased of ORC and its relationships to the functional form of the affinity for negatively supercoiled DNA3,4,14,15, indicating that super- ATP-DnaA filament (described in the accompanying work by coiling may be generally important for the initial engagement of Erzberger et al.21) and to the RFC structure. To that end, we origins. These observations suggest that bacterial initiators may provide have derived an EM reconstruction of the Drosophila melanogaster useful mechanistic insights into eukaryotic initiation, and vice versa. complex and tested the possible docking of these high- In all organisms and viruses, a key step in the pathway to initiation resolution X-ray models into the volume of the eukaryotic of DNA synthesis is the loading of a DNA helicase. In prokaryotic ORC structure. 1Division of Biochemistry & Molecular Biology, Molecular & Cell Biology Department, 1 Barker Hall, University of California, Berkeley, California 94720, USA. 2Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, California 94720, USA. 3Howard Hughes Medical Institute, Molecular and Cell Biology Department, LSA 355 #3200, University of California, Berkeley, California 94720-3200, USA. Correspondence should be addressed to M.B. ([email protected]) or E.N. ([email protected]). Received 14 April; accepted 21 June; published online 9 July 2006; doi:10.1038/nsmb1121 684 VOLUME 13 NUMBER 8 AUGUST 2006 NATURE STRUCTURAL & MOLECULAR BIOLOGY ARTICLES resolution of 34 A˚ , according to the 0.5 Fourier-shell correlation (FSC) abApo-ORC criterion (Fig. 1 and Supplementary Fig. 2 online) and shows an even coverage of angular space, without any apparent missing cone (Supplementary Fig. 2). From both the two-dimensional (Fig. 1b) and three-dimensional data (Fig. 1c), we observed that apo-ORC forms an elongated structure, with maximal dimensions of 170 A˚ Â 115 A˚ .Thethree- dimensional structure reveals a large major domain with distinct secondary features (Supplementary Video 1 online). The major domain forms the core of the complex and has a spiral-crescent shape that encompasses a B25-A˚ -wide channel. Density protruding off the top of this toroidal core results in an S-shaped molecule. This c 90° 90° 90° secondary density appears as a right-handed ‘boxing glove,’ where the ‘thumb’ contributes to the top of the channel and the ‘fingers’ curl back toward the major domain. Peripheral to the channel, at the base Channel 170 Å * of the complex, is a hole that exits through a small circular ‘collar’ Apo resting on the back of the core domain (Fig. 1c). Collar To visualize nucleotide-dependent changes in the ORC structure, we obtained images of the ATPg-S–bound form of ORC (Fig. 2a). Figure 1 EM analysis of Drosophila melanogaster ORC. (a) Electron Reference-free class averages (Fig. 2b) confirmed the expected simi- micrograph of negatively stained apo-ORC (scale bar ¼ 50 nm). (b) Examples of reference-free two-dimensional class averages. (c)34-A˚ larity between complexes with and without added nucleotide. We http://www.nature.com/nsmb resolution reconstruction of Drosophila ORC rendered as a gray isosurface. then produced a three-dimensional reconstruction of ATPg-S–bound 23 Each view represents a 901 counter-clockwise turn about the y-axis. Dashed ORC by iterative reference projection matching ,usingthe circle marks the collar. apocomplex as the initial reference (Fig. 2, Supplementary Fig. 2 and Supplementary Video 1). To avoid reference bias during projection matching, the initial apo-ORC reference volume was RESULTS filtered to 60 A˚ (Supplementary Fig. 3 online). The final ATP-ORC Nucleotide-dependent conformational changes in ORC structure, refined to 32-A˚ resolution, shows that the addition of In preparation for a structural analysis of Drosophila ORC, the nucleotide induces a conformational change that results in a tighten- recombinant protein was purified to homogeneity (Supplementary ing of the core spiral element (Fig. 2d, see below). In addition, changes Fig. 1 online). Drosophila ORC has emerged as a stable metazoan are observed in the collar, which opens in comparison to the apo Nature Publishing Group Group Nature Publishing ORC complex; unlike human ORC, all six subunits remain assembled structure (Fig. 2c,d). Concomitant with this change, thickening is 6 even in the absence of nucleotide, making it a more ideal complex observed in the mass that connects the uppermost subdomain to the 12 200 for structural studies . Electron micrographs of the negatively core. To verify that the differences between the two reconstructions © stained complex reveal that it is stable and does not aggregate on are substantial compared to the internal variability within each data the sample grid (Figs. 1 and 2). Tilt-pair data were collected from the set, three-dimensional variance maps were calculated for each recon- apocomplex for an ab initio reconstruction. The untilted particle data struction24,25. For both the apo and the ATP reconstruction, regions set was subjected to two-dimensional reference-free alignment and of high variance are very limited and localize mostly to the classification (Fig. 1b), and the tilted data was used to generate class stain-retaining cavities in the complex (Supplementary Fig. 4 online). volumes by the random conical tilt (RCT) method22. After merging A recent EM reconstruction of ATP-bound Saccharomyces cerevisiae together five class volumes, we used the resulting structure ORC26 also shows an elongated complex of similar dimensions, with a for projection-matching refinement. The final reconstruction has a ring domain resembling the collar in the Drosophila ORC reconstruc- tion. However, our structure is quite different in overall appearance from the
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