Significance: All cellular life forms use a ring-shaped hexameric helicase during DNA replication. CMG (Cdc45, Mcm2–7, GINS) is the eukaryotic replicative helicase. CMG contains the ring-shaped hexameric Mcm2–7 that harbors the helicase motors. CMG is known to bind many other proteins, including a leading and lagging polymerase and primase. Thus, the threading of DNA through the CMG helicase at a replication fork determines the orientation of the associated polymerases at the replication fork, an important structural feature with many consequences that may direct future experimentation. This report uses cryo-EM single-particle reconstruction to image CMG that motored to a block site at a forked junction, enabling direct visualization of DNA threading through CMG.

A 3.9-Å-resolution cryo-EM structure of the S. cerevisiae OCCM replicative helicase loading complex bound to DNA shows how ORC and Cdc6 recognize DNA origins, and reveals details of how the Mcm2–7 hexamer ring is loaded onto the DNA helix. To initiate DNA replication, the origin recognition complex (ORC) and Cdc6 load an Mcm2–7 double hexamer onto DNA. Without ATP hydrolysis, ORC–Cdc6 recruits one Cdt1-bound Mcm2–7 hexamer, thus forming an ORC–Cdc6–Cdt1–Mcm2–7 (OCCM) helicase-loading intermediate. Here we report a 3.9-Å structure of Saccharomyces cerevisiae OCCM on DNA. Flexible Mcm2–7 winged-helix domains (WHDs) engage ORC–Cdc6. A three-domain Cdt1 configuration embraces Mcm2, Mcm4, and Mcm6, thus comprising nearly half of the hexamer. The Cdt1 C-terminal domain extends to the Mcm6 WHD, which binds the Orc4 WHD. DNA passes through the ORC–Cdc6 and Mcm2–7 rings. Origin DNA interaction is mediated by an α-helix within Orc4 and positively charged loops within Orc2 and Cdc6. The Mcm2–7 C-tier AAA+ ring is topologically closed by an Mcm5 loop that embraces Mcm2, but the N-tier-ring Mcm2-Mcm5 interface remains open. This structure suggests a loading mechanism of the first Cdt1-bound Mcm2–7 hexamer by ORC–Cdc6.

The S. cerevisiae plasma membrane H⁺-ATPase, Pma1, is a P3A-type ATPase and the primary protein component of the membrane compartment of Pma1 (MCP). Like other plasma membrane H⁺-ATPases, Pma1 assembles and functions as a hexamer, a property unique to this subfamily among the larger family of P-type ATPases. It has been unclear how Pma1 organizes the yeast membrane into MCP microdomains, or why it is that Pma1 needs to assemble into a hexamer to establish the membrane electrochemical proton gradient. Here we report a high-resolution cryo-EM study of native Pma1 hexamers embedded in endogenous lipids. Remarkably, we found that the Pma1 hexamer encircles a liquid-crystalline membrane domain composed of 57 ordered lipid molecules. The Pma1-encircled lipid patch structure likely serves as the building block of the MCP. At pH 7.4, the carboxyl-terminal regulatory α-helix binds to the phosphorylation domains of two neighboring Pma1 subunits, locking the hexamer in the autoinhibited state. The regulatory helix becomes disordered at lower pH, leading to activation of the Pma1 hexamer. The activation process is accompanied by a 6.7 Å downward shift and a 40° rotation of transmembrane helices 1 and 2 that line the proton translocation path. The conformational changes have enabled us to propose a detailed mechanism for ATP-hydrolysis-driven proton pumping across the plasma membrane. Our structures will facilitate the development of antifungal drugs that target this essential protein.