DNA binding polarity, dimerization, and ATPase ring remodeling in the CMG helicase of the eukaryotic replisome
Alessandro Costa,
Ludovic Renault,
Paolo Swuec,
Tatjana Petojevic,
James J Pesavento,
Ivar Ilves,
Kirsty MacLellan-Gibson,
Roland A Fleck,
Michael R Botchan,
James M Berger
Affiliations
Alessandro Costa
London Research Institute, Cancer Research UK, London, United Kingdom
Ludovic Renault
London Research Institute, Cancer Research UK, London, United Kingdom; Department of Imaging, National Institute for Biological Standards and Control, Potters Bar, United Kingdom
Paolo Swuec
London Research Institute, Cancer Research UK, London, United Kingdom
Tatjana Petojevic
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
James J Pesavento
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
Ivar Ilves
Institute of Technology, Faculty of Science and Technology, University of Tartu, Tartu, Estonia
Kirsty MacLellan-Gibson
Department of Imaging, National Institute for Biological Standards and Control, Potters Bar, United Kingdom
Roland A Fleck
Department of Imaging, National Institute for Biological Standards and Control, Potters Bar, United Kingdom
Michael R Botchan
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
James M Berger
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, United States
The Cdc45/Mcm2-7/GINS (CMG) helicase separates DNA strands during replication in eukaryotes. How the CMG is assembled and engages DNA substrates remains unclear. Using electron microscopy, we have determined the structure of the CMG in the presence of ATPγS and a DNA duplex bearing a 3′ single-stranded tail. The structure shows that the MCM subunits of the CMG bind preferentially to single-stranded DNA, establishes the polarity by which DNA enters into the Mcm2-7 pore, and explains how Cdc45 helps prevent DNA from dissociating from the helicase. The Mcm2-7 subcomplex forms a cracked-ring, right-handed spiral when DNA and nucleotide are bound, revealing unexpected congruencies between the CMG and both bacterial DnaB helicases and the AAA+ motor of the eukaryotic proteasome. The existence of a subpopulation of dimeric CMGs establishes the subunit register of Mcm2-7 double hexamers and together with the spiral form highlights how Mcm2-7 transitions through different conformational and assembly states as it matures into a functional helicase.
