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

Abstract

Helicases are a class of motor proteins that move directionally along a nucleic acid phosphodiester backbone and separate two annealed nucleic acid strands by using ATP hydrolysis. Helicases exist in all living organisms. Based on their shared sequence motifs, they are classified into 6 classes in which superfamilies 1 and 2 are not ring‐shaped structure and superfamilies 3–6 are ring‐forming helicases. In eukaryotes, DNA helicase CMG consists of the Mcm2–7 hexameric ring along with five accessory factors. The Mcm2–7 heterohexamer, like all hexameric helicases, is shaped like a ring with two tiers referred to as an NTD tier (N‐terminal domain) and a CTD tier (C‐terminal domain); the CTD tier contains the ATP motors. In principle, either tier could translocate ahead of the other during movement on DNA. 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.Support or Funding InformationThis work was funded by NIH Grants GM111472 (to H.L.) and GM115809 (to M.E.O.); the Van Andel Research Institute (H.L); and the Howard Hughes Medical Institute (M.E.O.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.