Structure of Eukaryotic CMG Helicase at a Replication Fork and Implications for Replisome Architecture and Origin Initiation

Abstract

In eukaryotes, DNA replisome is a large protein complex that carries out DNA replication, which contains several enzymatic protein subunits, such as helicase, RNA primase and DNA polymerase. Replisome generates a DNA replication fork and duplicates both the leading and lagging strand of duplex DNA. Previous studies showed the Ctf4 trimer is proposed to be a hub to coordinate the subunits in replisome, Ctf4 trimer may link CMG and Pol a through binding CIP motif of Sld5 and Pol a. By using single‐particle cryo‐EM of an active CMG on the forked DNA with a dual biotin‐streptavidin block, we solved the first structure of helicase in complex with a DNA fork. The DNA duplex stem penetrates into the central channel of the NTD tier and the unwound leading ssDNA traverses the channel through the NTD tier into the CTD motor tier, 5′‐3′ through CMG. Therefore, during translocation the CTD motor is behind the NTD ring that is pushed ahead to face the fork, opposite the currently accepted polarity. The polarity of the NTD ahead of the CTD places the leading Polɛ below CMG and Polα‐primase at the top of CMG, above the unwinding point of the replication fork. The DNA direction requires that the Mcm rings must first pass one another when the Mcm2‐7 double hexamer come apart. Interestingly, the new NTD‐to‐CTD polarity of translocation reveals an unforeseen quality control mechanism that each Mcm2‐7 is fully converted from encircling dsDNA to ssDNA before they can depart from the origin. We propose this architecture of two motors head to head may generate energy that underlies initial melting at the origin.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.