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Nat Commun. 2018 Nov 29;9(1):5061. doi: 10.1038/s41467-018-07417-1.

Structure of DNA-CMG-Pol epsilon elucidates the roles of the non-catalytic polymerase modules in the eukaryotic replisome.

Author information

1
Macromolecular Machines Laboratory, The Francis Crick Institute, 1 Midland Road, London, NW1 1AT, UK.
2
Chromosome Replication Laboratory, The Francis Crick Institute, 1 Midland Road, London, NW1 1AT, UK.
3
Structural Biology Science Technology Platform, The Francis Crick Institute, 1 Midland Road, London, NW1 1AT, UK.
4
Department of Structural and Molecular Biology, Institute of Structural and Molecular Biology, University College London, London, UK.
5
Institute of Structural and Molecular Biology, Biological Sciences, Birkbeck College, London, WC1E 7HX, UK.
6
Macromolecular Machines Laboratory, The Francis Crick Institute, 1 Midland Road, London, NW1 1AT, UK. alessandro.costa@crick.ac.uk.

Abstract

Eukaryotic origin firing depends on assembly of the Cdc45-MCM-GINS (CMG) helicase. A key step is the recruitment of GINS that requires the leading-strand polymerase Pol epsilon, composed of Pol2, Dpb2, Dpb3, Dpb4. While a truncation of the catalytic N-terminal Pol2 supports cell division, Dpb2 and C-terminal Pol2 (C-Pol2) are essential for viability. Dpb2 and C-Pol2 are non-catalytic modules, shown or predicted to be related to an exonuclease and DNA polymerase, respectively. Here, we present the cryo-EM structure of the isolated C-Pol2/Dpb2 heterodimer, revealing that C-Pol2 contains a DNA polymerase fold. We also present the structure of CMG/C-Pol2/Dpb2 on a DNA fork, and find that polymerase binding changes both the helicase structure and fork-junction engagement. Inter-subunit contacts that keep the helicase-polymerase complex together explain several cellular phenotypes. At least some of these contacts are preserved during Pol epsilon-dependent CMG assembly on path to origin firing, as observed with DNA replication reconstituted in vitro.

PMID:
30498216
PMCID:
PMC6265327
DOI:
10.1038/s41467-018-07417-1
[Indexed for MEDLINE]
Free PMC Article

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