Structural basis of Mcm2–7 replicative helicase loading by ORC–Cdc6 and Cdt1

Abstract

To start DNA replication, the Origin Recognition Complex (ORC) and Cdc6 load a Mcm2-7 double hexamer onto DNA. Without ATP hydrolysis, ORC-Cdc6 recruits one Cdt1-bound Mcm2-7 hexamer, forming an ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) helicase loading intermediate. Here we report a 3.9Å structure of the OCCM on DNA. Flexible Mcm2-7 winged-helix domains (WHD) engage ORC-Cdc6. A three-domain Cdt1 configuration embraces Mcm2, Mcm4, and Mcm6, nearly half of the hexamer. The Cdt1 C-terminal domain extends to the Mcm6 WHD, which binds Orc4 WHD. DNA passes through the ORC-Cdc6 and Mcm2-7 rings. Origin DNA interaction is mediated by an α-helix in Orc4 and positively charged loops in Orc2 and Cdc6. The Mcm2-7 C-tier AAA+ ring is topologically closed by a Mcm5 loop that embraces Mcm2, but the N-tier ring Mcm2-Mcm5 interface remains open. This structure suggests loading mechanics of the first Cdt1-bound Mcm2-7 hexamer by ORC-Cdc6.

From structure to mechanism—understanding initiation of DNA replication

“…Unlike other Orc/Cdc6 proteins, Orc4 appears responsible for sequence-specific DNA interactions in the OCCM, as it uniquely makes base-specific contacts in the major groove of the DNA ( Fig. 2D; Yuan et al 2017). However, the functional relevance of these interactions is still outstanding.…”

“…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.…”

“…In this context, Orc2, Orc4, and Cdc6 make direct DNA contacts, while Orc1, Orc3, and Orc5 do not touch the DNA ( Fig. 2B; Yuan et al 2017). Within the Orc2 protein, only the ISM of the AAA + domain interacts with DNA, mostly contacting the phosphate backbone (Fig.…”

“…1A;Bell and Stillman 1992). This complex consists of six subunits and is organized in a C shape, with DNA being inserted into the central cleft, allowing for multiple protein-DNA contacts (Lee and Bell 1997;Speck et al 2005;Sun et al 2012;Yuan et al 2017). During late M phase of the cell cycle, Cdc6 binding to an ORC/origin DNA complex starts a process of regulated protein assembly that culminates in the formation of the replication fork in S phase (Fig.…”