Structure of the active form of human origin recognition complex and its ATPase motor module

Abstract

Binding of the Origin Recognition Complex (ORC) to origins of replication marks the first step in the initiation of replication of the genome in all eukaryotic cells. Here, we report the structure of the active form of human ORC determined by X-ray crystallography and cryo-electron microscopy. The complex is composed of an ORC1/4/5 motor module lobe in an organization reminiscent of the DNA polymerase clamp loader complexes. A second lobe contains the ORC2/3 subunits. The complex is organized as a double-layered shallow corkscrew, with the AAA+ and AAA+-like domains forming one layer, and the winged-helix domains (WHDs) forming a top layer. CDC6 fits easily between ORC1 and ORC2, completing the ring and the DNA-binding channel, forming an additional ATP hydrolysis site. Analysis of the ATPase activity of the complex provides a basis for understanding ORC activity as well as molecular defects observed in Meier-Gorlin Syndrome mutations.DOI: http://dx.doi.org/10.7554/eLife.20818.001

From structure to mechanism—understanding initiation of DNA replication

“…Remarkably, this Orc6 helix is mutated in Meier-Gorlin syndrome. Indeed, this disease results in primordial dwarfism in humans, which is also caused by mutations in Orc1, Orc4, Cdt1, and Cdc6 (Bicknell et al 2011;Tocilj et al 2017). Consistently, in the case of Orc6, the mutation results in a defective Orc3 interaction, which in turn was shown to reduce MCM2-7 loading in Drosophila (Bleichert et al 2013).…”

“…Crystallography and cryo-EM have generated near-atomic resolution structures of several large protein complexes, including Drosophila melanogaster and Homo sapiens ORC (Bleichert et al 2015;Tocilj et al 2017); Saccharomyces cerevisiae MCM2-7 and MCM2-7/Cdt1 (Zhai et al 2017), OCCM complex , and MCM2-7 DH ; S. cerevisiae and D. melanogaster CMG (Abid Ali et al 2016;Yuan et al 2016;Georgescu et al 2017); S. cerevisiae Ctf4 trimer (Simon et al 2014), polymerase ε (Hogg et al 2014), and polymerase δ (Swan et al 2009); and H. sapiens Mcm2-H3/H4 (Huang et al 2015;Richet et al 2015). Here we concentrate on four key complexes involved in initiation of DNA replication; namely, S. cerevisiae ORC, OCCM, MCM2-7 DH, and CMG (Fig.…”

“…Orc1-5, but not Orc6, have a conserved protein structure consisting of one N-terminal AAA + domain and one C-terminal winged helix domain (WHD) ( Fig. 2A; Bleichert et al 2015;Tocilj et al 2017;Yuan et al 2017). Structural analysis showed that H. sapiens Orc6 has homology with transcription factor TFIIB (Liu et al 2011).…”

“…However, how the Cdc6 ATPase-dependent regulation of sequence specificity works (Speck and Stillman 2007) is still unknown at a structural level. The crystal structure of the D. melanogaster ORC core revealed that D. melanogaster Orc3-4-5 has a configuration similar to that of S. cerevisiae and H. sapiens Orc3-4-5, but the whole structure adopts an autoinhibited conformation that is incompatible with DNA and Cdc6 binding (Bleichert et al 2015;Tocilj et al 2017;Yuan et al 2017). Aligning the D. melanogaster ORC structure to the S. cerevisiae ORC structure shows that the D. melanogaster Orc1 AAA + domain and the D. melanogaster Orc2 WHD cover the central channel of the ORC DNA passage ( Fig.…”

Structural insight into the unique dsDNA binding topology of the human ORC2 wing helix domain

“…The crystal structure of the Drosophila Orc1‐6 complex reveals that the six subunits form a tight complex and that Orc2 interacts extensively with Orc3 . The Orc2–Orc3 architecture is also conserved in human, as confirmed in the recent low‐resolution cryo‐EM heterodimer of human ORC2–ORC3 (PDB code: http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5UJ8) . WHD is a common nucleic‐acid‐binding element found in all kingdoms of life .…”