Abstract
High-resolution structures have not been reported for replicative helicases at a replication fork at atomic resolution, a prerequisite to understanding the unwinding mechanism. The eukaryotic replicative CMG (Cdc45, Mcm2-7, GINS) helicase contains a Mcm2-7 motor ring, with the N-tier ring in front and the C-tier motor ring behind. The N-tier ring is structurally divided into a zinc finger (ZF) sub-ring followed by the oligosaccharide/oligonucleotide-binding (OB) fold ring. Here we report the cryo-EM structure of CMG on forked DNA at 3.9 Å, revealing that parental DNA enters the ZF sub-ring and strand separation occurs at the bottom of the ZF sub-ring, where the lagging strand is blocked and diverted sideways by OB hairpin-loops of Mcm3, Mcm4, Mcm6, and Mcm7. Thus, instead of employing a specific steric exclusion process, or even a separation pin, unwinding is achieved via a “dam-and-diversion tunnel” mechanism that does not require specific protein-DNA interaction. The C-tier motor ring contains spirally configured PS1 and H2I loops of Mcms 2, 3, 5, 6 that translocate on the spirally-configured leading strand, and thereby pull the preceding DNA segment through the diversion tunnel for strand separation.
Highlights
High-resolution structures have not been reported for replicative helicases at a replication fork at atomic resolution, a prerequisite to understanding the unwinding mechanism
The pre-sensor 1 (PS1) loops are proposed to pull the single-stranded DNA (ssDNA) such that the parental duplex splits at the top of the helicase, a process known as the steric exclusion mechanism of helicase action, because one strand is excluded from the central channel while the other strand is pulled through the central channel[17]
Our earlier study of the budding yeast S. cerevisiae CMG bound to a forked DNA demonstrated that the parental duplex enters the N-tier of CMG a short distance[19], which was different from the classic steric exclusion models[2,7]
Summary
High-resolution structures have not been reported for replicative helicases at a replication fork at atomic resolution, a prerequisite to understanding the unwinding mechanism. Details of how steric exclusion splits the strands rather than bringing them both into the central channel of the helicase are unknown, as high-resolution structures of helicases with bound forked DNA are lacking. There was a long density gap between the parental dsDNA region in the Ntier and the leading strand DNA in the C-tier motor ring; in other words, the DNA fork junction region was not visualized in that study. The primary motivation of this manuscript is to visualize the forked DNA junction structure at atomic or near atomic resolution—high enough resolution to define a precise mechanism of dsDNA unwinding by a eukaryotic helicase performing steric exclusion, that could otherwise draw both DNA strands through the central channel. We analyzed a CMG–Mcm[10] complex because Mcm[10] binds CMG tightly and can be isolated as a CMG–Mcm[10] complex[20], and because Mcm[10] contains a DNA-binding module that likely underies its observed ability to facilitate DNA binding of CMG-Mcm1021–25
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