Abstract
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, 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.
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Acknowledgements
We thank M. Gossen at the Max Delbruck Institute for numerous gifts of the Drosophila Orc5 monoclonal antibody, D. Remus for helpful discussions and the Office of Biological and Environmental Research of the US Department of Energy for support of E.N. E.N. is a Howard Hughes Medical Institute Investigator. This work was supported by US National Institutes of Health grant R39 CA 30490 to M.B.
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Contributions
M.G.C. contributed ORC sample preparation and EM analysis; J.P.E. contributed DnaA docking and sequence alignments; P.G. contributed three-dimensional variance analysis; A.E.L. contributed computational assistance; J.M.B. contributed ORC cartoon model, docking supervision and DnaA mass/volume calculations; E.N. supervised EM analysis and contributed to the manuscript; M.B. supervised biochemistry and contributed to the manuscript.
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Supplementary information
Supplementary Fig. 1
ORC purification. (PDF 212 kb)
Supplementary Fig. 2
Fourier shell correlation and angular distribution of ORC reconstructions. (PDF 186 kb)
Supplementary Fig. 3
Reference projections for ATP-ORC reconstruction. (PDF 1018 kb)
Supplementary Fig. 4
Three-dimensional variance of ORC reconstructions. (PDF 1883 kb)
Supplementary Video 1
EM reconstructions of apo- and ATP-ORC. (MOV 936 kb)
Supplementary Video 2
Docking of ATP-DnaA pentamer into apo-ORC. (MOV 2500 kb)
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Clarey, M., Erzberger, J., Grob, P. et al. Nucleotide-dependent conformational changes in the DnaA-like core of the origin recognition complex. Nat Struct Mol Biol 13, 684–690 (2006). https://doi.org/10.1038/nsmb1121
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DOI: https://doi.org/10.1038/nsmb1121
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