Abstract
Eukaryotic DNA replication initiation relies on the origin recognition complex (ORC), a DNA-binding ATPase that loads the Mcm2–7 replicative helicase onto replication origins. Here, we report cryo-electron microscopy (cryo-EM) structures of DNA-bound Drosophila ORC with and without the co-loader Cdc6. These structures reveal that Orc1 and Orc4 constitute the primary DNA binding site in the ORC ring and cooperate with the winged-helix domains to stabilize DNA bending. A loop region near the catalytic Walker B motif of Orc1 directly contacts DNA, allosterically coupling DNA binding to ORC’s ATPase site. Correlating structural and biochemical data show that DNA sequence modulates DNA binding and remodeling by ORC, and that DNA bending promotes Mcm2–7 loading in vitro. Together, these findings explain the distinct DNA sequence-dependencies of metazoan and S. cerevisiae initiators in origin recognition and support a model in which DNA geometry and bendability contribute to Mcm2–7 loading site selection in metazoans.
Highlights
Eukaryotic DNA replication initiation relies on the origin recognition complex (ORC), a DNAbinding ATPase that loads the Mcm[2,3,4,5,6,7] replicative helicase onto replication origins
Through testing of various deletion constructs of ORC and Cdc[6], we found that removal of the N-terminal regions of Orc[1] (Orc1ΔN) and Cdc[6] (Cdc6ΔN) improves solubility in the presence of DNA and monodispersity of the sample after freezing
DmOrc[1], DmCdc[6], and DmCdt[1] proteins lacking N-terminal, intrinsically disordered regions (IDRs) loaded Mcm[2,3,4,5,6,7] onto DNA to similar extents as full-length proteins (Fig. 1a, b). These findings indicate that the truncated DmORC and DmCdc[6] constructs are functional and that the IDRs in Orc[1], Cdc[6], and Cdt[1] are not essential for Mcm[2,3,4,5,6,7] loading in vitro
Summary
Eukaryotic DNA replication initiation relies on the origin recognition complex (ORC), a DNAbinding ATPase that loads the Mcm[2,3,4,5,6,7] replicative helicase onto replication origins. We report cryo-electron microscopy (cryo-EM) structures of DNA-bound Drosophila ORC with and without the co-loader Cdc[6] These structures reveal that Orc[1] and Orc[4] constitute the primary DNA binding site in the ORC ring and cooperate with the winged-helix domains to stabilize DNA bending. Correlating structural and biochemical data show that DNA sequence modulates DNA binding and remodeling by ORC, and that DNA bending promotes Mcm[2,3,4,5,6,7] loading in vitro. Structural studies have revealed that the initiator binds DNA in the center of the ORC ring, bending DNA to prepare origins for Mcm[2,3,4,5,6,7] recruitment and loading[18,20,21]. Whether DNA remodeling is required for Mcm[2,3,4,5,6,7] loading is likewise uncertain
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