title: DNA binding properties of human Cdc45 suggest a function as molecular wedge for DNA unwinding.
abstract: The cell division cycle protein 45 (Cdc45) represents an essential replication factor that, together with the Mcm2-7 complex and the four subunits of GINS, forms the replicative DNA helicase in eukaryotes. Recombinant human Cdc45 (hCdc45) was structurally characterized and its DNA-binding properties were determined. Synchrotron radiation circular dichroism spectroscopy, dynamic light scattering, small-angle X-ray scattering and atomic force microscopy revealed that hCdc45 exists as an alpha-helical monomer and possesses a structure similar to its bacterial homolog RecJ. hCdc45 bound long (113-mer or 80-mer) single-stranded DNA fragments with a higher affinity than shorter ones (34-mer). hCdc45 displayed a preference for 3' protruding strands and bound tightly to single-strand/double-strand DNA junctions, such as those presented by Y-shaped DNA, bubbles and displacement loops, all of which appear transiently during the initiation of DNA replication. Collectively, our findings suggest that hCdc45 not only binds to but also slides on DNA with a 3'-5' polarity and, thereby acts as a molecular 'wedge' to initiate DNA strand displacement.
authors.affiliation: Research Group Biochemistry, Leibniz Institute for Age Research -Fritz Lipmann Institute, Beutenbergstrasse 11, D-07745 Jena, Germany, Laboratory of Molecular Biology IBB PAS, Affiliated with University of Gdansk, Wita Stwosza 59 Gdansk, Poland, Rudolf Virchow Center, DFG Research Center for Experimental Biomedicine, Josef Schneider Strasse 2, 7080 Wurzburg, Germany, Department of Biochemistry, Oulu, P.O. Box 3000, University of Oulu, Oulu 90014, Finland, Department of Chemistry, University of Hamburg/DESY, Notkestrasse 85, 22607 Hamburg, Germany, Biocenter Oulu, P.O. Box 3000, University of Oulu, Oulu 90014, Finland and Center for Molecular Biomedicine, Friedrich-Schiller University, Biochemistry Department, Jena, Germany.
authors.forename: Anna
authors.initials: A
authors.lastname: Szambowska
authors.forename: Ingrid
authors.initials: I
authors.lastname: Tessmer
authors.forename: Petri
authors.initials: P
authors.lastname: Kursula
authors.forename: Christian
authors.initials: C
authors.lastname: Usskilat
authors.forename: Piotr
authors.initials: P
authors.lastname: Prus
authors.forename: Helmut
authors.initials: H
authors.lastname: Pospiech
authors.forename: Frank
authors.initials: F
authors.lastname: Grosse
mesh_terms: D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D017510:Protein Folding; D017433:Protein Structure, Secondary
chemical_list: C113571:CDC45 protein, human; D018797:Cell Cycle Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D004247:DNA