Structure of eukaryotic CMG helicase at a replication fork and implications to replisome architecture and origin initiation

The eukaryotic CMG (Cdc45, Mcm2–7, GINS) helicase consists of the Mcm2–7 hexameric ring along with five accessory factors. The Mcm2–7 heterohexamer, like other hexameric helicases, is shaped like a ring with two tiers, an N-tier ring composed of the N-terminal domains, and a C-tier of C-terminal dom...

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Published in:Proceedings of the National Academy of Sciences - PNAS 2017-01, Vol.114 (5), p.E697-E706
Main Authors: Georgescu, Roxana, Yuan, Zuanning, Bai, Lin, de Luna Almeida Santos, Ruda, Sun, Jingchuan, Zhang, Dan, Yurieva, Olga, Li, Huilin, O’Donnell, Michael E.
Format: Article
Language:English
Subjects:
Biological Sciences
Deoxyribonucleic acid
DNA
DNA Replication
DNA, Single-Stranded - chemistry
DNA-Binding Proteins - chemistry
Eukaryotes
Minichromosome Maintenance Proteins - chemistry
Models, Molecular
Molecules
Nuclear Proteins - chemistry
PNAS Plus
Protein Conformation
Replication Origin
Saccharomyces cerevisiae Proteins - chemistry
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Summary:The eukaryotic CMG (Cdc45, Mcm2–7, GINS) helicase consists of the Mcm2–7 hexameric ring along with five accessory factors. The Mcm2–7 heterohexamer, like other hexameric helicases, is shaped like a ring with two tiers, an N-tier ring composed of the N-terminal domains, and a C-tier of C-terminal domains; the C-tier contains the motor. In principle, either tier could translocate ahead of the other during movement on DNA. We have used cryo-EMsingle-particle 3D reconstruction to solve the structure of CMG in complex with a DNA fork. The duplex stem penetrates into the central channel of the N-tier and the unwound leading single-strand DNA traverses the channel through the N-tier into the C-tier motor, 5′-3′ through CMG. Therefore, the N-tier ring is pushed ahead by the C-tier ring during CMG translocation, opposite the currently accepted polarity. The polarity of the N-tier ahead of the C-tier places the leading Pol ε below CMG and Pol α-primase at the top of CMG at the replication fork. Surprisingly, the new N-tier to C-tier polarity of translocation reveals an unforeseen quality-control mechanism at the origin. Thus, upon assembly of head-to-head CMGs that encircle double-stranded DNA at the origin, the two CMGs must pass one another to leave the origin and both must remodel onto opposite strands of single-stranded DNA to do so. We propose that head-to-head motors may generate energy that underlies initialmelting at the origin.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.1620500114