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.
Highlights
The first step in genome replication, the binding of the Origin Recognition Complex (ORC) at origins of DNA replication, triggers a series of highly coordinated steps leading to the assembly of pre-replicative complexes in a process that involves CDC6 binding to ORC (Bell and Labib, 2016)
Each ORC subunit is comprised of three domains – the RecA-fold, the a-helical lid and the a-helical winged-helix domain (WHD), the winged-helix domains (WHDs) domain was truncated in ORC5
The structure of HsORC is substantially different from the known Drosophila melanogaster ORC (DmORC) structure, it is very similar to the cryo-electron microscopy (cryoEM) structure of ORC from Saccharomyces cerevisiae (ScORC) observed in the context of the ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) complex (Sun et al, 2014; Yuan, Riera, Bai, Sun, Nandi, Spanos, Chen, Barbon, Rappsilber, Stillman, Speck, and Li, personal communication)
Summary
The first step in genome replication, the binding of the Origin Recognition Complex (ORC) at origins of DNA replication, triggers a series of highly coordinated steps leading to the assembly of pre-replicative complexes (pre-RCs) in a process that involves CDC6 binding to ORC (Bell and Labib, 2016). ORC and CDC6 function as an ATP-dependent assembler that first recruits a ring-shaped MCM2-7 hexamer with bound Cdt to DNA, and loads a second MCM2-7 hexamer in a headto-head orientation, whereby this double hexamer is topologically linked to double-stranded DNA (reviewed in [O’Donnell et al, 2013; Siddiqui et al, 2013; Bell and Labib, 2016]). These MCM2-7 double hexamers mark the location of potential origins of DNA replication that can be activated during S phase to replicate the genome. Orc and Orc are bona-fide members of a large class of AAA+ proteins that contain the characteristic Walker-A and Walker-B motifs, but the only active ATPase in S. cerevisiae
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