title: Cdc45 (cell division cycle protein 45) guards the gate of the Eukaryote Replisome helicase stabilizing leading strand engagement.
abstract: DNA replication licensing is now understood to be the pathway that leads to the assembly of double hexamers of minichromosome maintenance (Mcm2-7) at origin sites. Cell division control protein 45 (Cdc45) and GINS proteins activate the latent Mcm2-7 helicase by inducing allosteric changes through binding, forming a Cdc45/Mcm2-7/GINS (CMG) complex that is competent to unwind duplex DNA. The CMG has an active gate between subunits Mcm2 and Mcm5 that opens and closes in response to nucleotide binding. The consequences of inappropriate Mcm2/5 gate actuation and the role of a side channel formed between GINS/Cdc45 and the outer edge of the Mcm2-7 ring for unwinding have remained unexplored. Here we uncover a novel function for Cdc45. Cross-linking studies trace the path of the DNA with the CMG complex at a fork junction between duplex and single strands with the bound CMG in an open or closed gate conformation. In the closed state, the lagging strand does not pass through the side channel, but in the open state, the leading strand surprisingly interacts with Cdc45. Mutations in the recombination protein J fold of Cdc45 that ablate this interaction diminish helicase activity. These data indicate that Cdc45 serves as a shield to guard against occasional slippage of the leading strand from the core channel.
authors.affiliation: Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley, CA 94720; Department of Biology, Chemistry and Pharmacy, Institute of Chemistry and Biochemistry, Freie Universität Berlin, 14195 Berlin, Germany;
authors.forename: Tatjana
authors.initials: T
authors.lastname: Petojevic
authors.affiliation: Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley, CA 94720;
authors.forename: James J
authors.initials: JJ
authors.lastname: Pesavento
authors.affiliation: Clare Hall Laboratories, London Research Institute, South Mimms, Herts EN6 3LD, United Kingdom; and.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Costa
authors.affiliation: Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley, CA 94720;
authors.forename: Jingdan
authors.initials: J
authors.lastname: Liang
authors.affiliation: Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley, CA 94720;
authors.forename: Zhijun
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Department of Biophysics and Biophysical Chemistry, School of Medicine, Johns Hopkins University, Baltimore, MD 21205 mbotchan@berkeley.edu jmberger@jhmi.edu.
authors.forename: James M
authors.initials: JM
authors.lastname: Berger
authors.affiliation: Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley, CA 94720; mbotchan@berkeley.edu jmberger@jhmi.edu.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Botchan
mesh_terms: D000595:Amino Acid Sequence; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000502:physiology; D004247:DNA / Q000378:metabolism; D004265:DNA Helicases / Q000378:metabolism*; D005456:Fluorescent Dyes; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid
chemical_list: D018797:Cell Cycle Proteins; D005456:Fluorescent Dyes; D004247:DNA; D004265:DNA Helicases
keywords: CMG helicase; Cdc45; DNA replication; Mcm2/5 gate; RecJ fold