title: Recruitment of Mcm10 to Sites of Replication Initiation Requires Direct Binding to the Minichromosome Maintenance (MCM) Complex.
abstract: Mcm10 is required for the initiation of eukaryotic DNA replication and contributes in some unknown way to the activation of the Cdc45-MCM-GINS (CMG) helicase. How Mcm10 is localized to sites of replication initiation is unclear, as current models indicate that direct binding to minichromosome maintenance (MCM) plays a role, but the details and functional importance of this interaction have not been determined. Here, we show that purified Mcm10 can bind both DNA-bound double hexamers and soluble single hexamers of MCM. The binding of Mcm10 to MCM requires the Mcm10 C terminus. Moreover, the binding site for Mcm10 on MCM includes the Mcm2 and Mcm6 subunits and overlaps that for the loading factor Cdt1. Whether Mcm10 recruitment to replication origins depends on CMG helicase assembly has been unclear. We show that Mcm10 recruitment occurs via two modes: low affinity recruitment in the absence of CMG assembly ("G1-like") and high affinity recruitment when CMG assembly takes place ("S-phase-like"). Mcm10 that cannot bind directly to MCM is defective in both modes of recruitment and is unable to support DNA replication. These findings indicate that Mcm10 is localized to replication initiation sites by directly binding MCM through the Mcm10 C terminus.
authors.affiliation: From The Francis Crick Institute, Clare Hall Laboratory, South Mimms, Hertfordshire EN6 3LD, United Kingdom.
authors.forename: Max E
authors.initials: ME
authors.lastname: Douglas
authors.affiliation: From The Francis Crick Institute, Clare Hall Laboratory, South Mimms, Hertfordshire EN6 3LD, United Kingdom. Electronic address: john.diffley@crick.ac.uk.
authors.forename: John F X
authors.initials: JFX
authors.lastname: Diffley
mesh_terms: D004261:DNA Replication; D064151:Minichromosome Maintenance Complex Component 6 / Q000378:metabolism*; D064110:Minichromosome Maintenance Proteins / Q000378:metabolism*; D011485:Protein Binding; D018741:Replication Origin; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*
chemical_list: C076365:MCM10 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C069199:MCM2 protein, S cerevisiae; C491207:MCM6 protein, S cerevisiae; D064151:Minichromosome Maintenance Complex Component 6; D064110:Minichromosome Maintenance Proteins
keywords: DNA helicase; DNA polymerase; DNA replication; DNA synthesis; mcm10; protein-DNA interaction; replication origin