Crystal structure of the eukaryotic origin recognition complex

Abstract

Initiation of cellular DNA replication is tightly controlled to sustain genomic integrity. In eukaryotes, the heterohexameric origin recognition complex (ORC) is essential for coordinating replication onset. The 3.5 Å resolution crystal structure of Drosophila ORC reveals that the 270 kDa initiator core complex comprises a two-layered notched ring in which a collar of winged-helix domains from the Orc1-5 subunits sits atop a layer of AAA+ ATPase folds. Although canonical inter-AAA+ domain interactions exist between four of the six ORC subunits, unanticipated features are also evident, including highly interdigitated domain-swapping interactions between the winged-helix folds and AAA+ modules of neighboring protomers, and a quasi-spiral arrangement of DNA binding elements that circumnavigate a ~20 Å wide channel in the center of the complex. Comparative analyses indicate that ORC encircles DNA, using its winged-helix domain face to engage the MCM2-7 complex during replicative helicase loading; however, an observed >90° out-of-plane rotation for the Orc1 AAA+ domain disrupts interactions with catalytic amino acids in Orc4, narrowing and sealing off entry into the central channel. Prima facie, our data indicate that Drosophila ORC can switch between active and autoinhibited conformations, suggesting a novel means for cell cycle and/or developmental control of ORC functions.

Orc1 Binding to Mitotic Chromosomes Precedes Spatial Patterning during G1 Phase and Assembly of the Origin Recognition Complex in Human Cells

“…Thus, motif I plays a dual role in Orc1, for mitotic chromosome association, and as part of the AAA ϩ module. Motif II, however, is present in a region of the AAA ϩ domain of Orc1 that protrudes out of the Orc1 structure present in a recent crystal structure of ORC from Drosophila (86). The structure of this Drosophila ORC, particularly Orc1, is in a conformation that would not allow ATPase activity and thus is most likely not the conformation of ORC that recruits Cdc6 and pre-RC proteins.…”

From structure to mechanism—understanding initiation of DNA replication

“…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).…”

“…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.…”

Profile of James M. Berger

“…Such a collaborative effort occurred in 2015, when Berger and colleagues Franziska Bleichart and Michael Botchan studied the origin recognition complex (ORC), a protein complex that directs DNA replication (9). Using the Argonne National Laboratory's powerful X-ray beam and gathering data from a beamline supported by the National Institute for General Medicine and the National Cancer Institute, the researchers obtained the first atomic-level insight into the ORC.…”