Structural basis of Mcm2-7 replicative helicase loading by ORC-Cdc6 and Cdt1

Zuanning Yuan; Alberto Riera; Lin Bai; Jingchuan Sun; Saikat Nandi; Christos Spanos; Zhuo Angel Chen; Marta Barbon; Juri Rappsilber; Bruce Stillman; Christian Speck; Huilin Li

doi:10.1038/nsmb.3372

Structural basis of Mcm2-7 replicative helicase loading by ORC-Cdc6 and Cdt1

Nat Struct Mol Biol. 2017 Mar;24(3):316-324. doi: 10.1038/nsmb.3372. Epub 2017 Feb 13.

Authors

Zuanning Yuan¹, Alberto Riera^{2

3}, Lin Bai¹, Jingchuan Sun¹, Saikat Nandi⁴, Christos Spanos⁵, Zhuo Angel Chen⁵, Marta Barbon^{2

3}, Juri Rappsilber^{5

6}, Bruce Stillman⁴, Christian Speck^{2

3}, Huilin Li¹

Affiliations

¹ Cryo-EM Structural Biology Laboratory, Van Andel Research Institute, Grand Rapids, Michigan, USA.
² MRC London Institute of Medical Sciences (LMS), London, UK.
³ DNA Replication Group, Institute of Clinical Sciences (ICS), Faculty of Medicine, Imperial College London, London, UK.
⁴ Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA.
⁵ Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, UK.
⁶ Chair of Bioanalytics, Institute of Biotechnology, Technische Universität Berlin, Berlin, Germany.

Abstract

To initiate DNA replication, the origin recognition complex (ORC) and Cdc6 load an Mcm2-7 double hexamer onto DNA. Without ATP hydrolysis, ORC-Cdc6 recruits one Cdt1-bound Mcm2-7 hexamer, thus forming an ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) helicase-loading intermediate. Here we report a 3.9-Å structure of Saccharomyces cerevisiae OCCM on DNA. Flexible Mcm2-7 winged-helix domains (WHDs) engage ORC-Cdc6. A three-domain Cdt1 configuration embraces Mcm2, Mcm4, and Mcm6, thus comprising nearly half of the hexamer. The Cdt1 C-terminal domain extends to the Mcm6 WHD, which binds the Orc4 WHD. DNA passes through the ORC-Cdc6 and Mcm2-7 rings. Origin DNA interaction is mediated by an α-helix within Orc4 and positively charged loops within Orc2 and Cdc6. The Mcm2-7 C-tier AAA+ ring is topologically closed by an Mcm5 loop that embraces Mcm2, but the N-tier-ring Mcm2-Mcm5 interface remains open. This structure suggests a loading mechanism of the first Cdt1-bound Mcm2-7 hexamer by ORC-Cdc6.

MeSH terms

Binding Sites
Cell Cycle Proteins / chemistry*
Cell Cycle Proteins / metabolism
Cell Cycle Proteins / ultrastructure
Cryoelectron Microscopy
DNA Replication*
DNA, Fungal / chemistry
DNA, Fungal / metabolism
DNA-Binding Proteins / chemistry*
DNA-Binding Proteins / metabolism
DNA-Binding Proteins / ultrastructure
Mass Spectrometry
Minichromosome Maintenance Proteins / chemistry*
Minichromosome Maintenance Proteins / metabolism
Minichromosome Maintenance Proteins / ultrastructure
Models, Molecular
Nucleotides / metabolism
Protein Binding
Protein Domains
Protein Multimerization
Protein Structure, Secondary
Replication Origin*
Saccharomyces cerevisiae / metabolism*
Saccharomyces cerevisiae Proteins / chemistry*
Saccharomyces cerevisiae Proteins / metabolism
Saccharomyces cerevisiae Proteins / ultrastructure

Substances

CDC6 protein, S cerevisiae
Cell Cycle Proteins
DNA, Fungal
DNA-Binding Proteins
Nucleotides
Saccharomyces cerevisiae Proteins
TAH11 protein, S cerevisiae
Minichromosome Maintenance Proteins

Abstract

MeSH terms

Substances

Grants and funding