Abstract
Genome replication is initiated from specific origin sites established by dynamic events. The Origin Recognition Complex (ORC) is necessary for orchestrating the initiation process by binding to origin DNA, recruiting CDC6, and assembling the MCM replicative helicase on DNA. Here we report five cryoEM structures of the human ORC (HsORC) that illustrate the native flexibility of the complex. The absence of ORC1 revealed a compact, stable complex of ORC2-5. Introduction of ORC1 opens the complex into several dynamic conformations. Two structures revealed dynamic movements of the ORC1 AAA+ and ORC2 winged-helix domains that likely impact DNA incorporation into the ORC core. Additional twist and pinch motions were observed in an open ORC conformation revealing a hinge at the ORC5·ORC3 interface that may facilitate ORC binding to DNA. Finally, a structure of ORC was determined with endogenous DNA bound in the core revealing important differences between human and yeast origin recognition.
Highlights
DNA replication is essential to all forms of life
Two HsORC constructs were generated for recombinant expression based on our previous study (Tocilj et al, 2017) with the following changes: ORC2 was extended to full-length, the StrepTag was moved from the N-terminus of ORC1 to the N-terminus of ORC3 to improve sample purity during affinity purification, and the Sumo tag on ORC1 was removed
One construct consisted of full-length ORC1, but after expression and purification, ORC1 was absent yielding a complex of ORC2-5
Summary
DNA replication is essential to all forms of life. In eukaryotes, replication is initiated from multiple start sites on chromosomes called replication origins (reviewed in Bell and Labib, 2016; Bleichert et al, 2017; Leonard and Mechali, 2013; On et al, 2018; Prioleau and MacAlpine, 2016). The very first step of this initiation process is accomplished by DNA association with the Origin Recognition Complex (ORC), a six-subunit protein that forms a partial ring around origin DNA (Li et al, 2018; Yuan et al, 2017). ORC must perform several tasks sequentially: bind DNA, find origin sites, and dissociate from DNA upon completion of the MCM double-hexamer loading and assembly. Single molecule studies have shown that ORC is released from DNA immediately following stable association of two MCM molecules with the DNA substrate (Ticau et al, 2015). These dynamic events likely require significant conformational changes in ORC
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