title: Cdc45-induced loading of human RPA onto single-stranded DNA.
abstract: Cell division cycle protein 45 (Cdc45) is an essential component of the eukaryotic replicative DNA helicase. We found that human Cdc45 forms a complex with the single-stranded DNA (ssDNA) binding protein RPA. Moreover, it actively loads RPA onto nascent ssDNA. Pull-down assays and surface plasmon resonance studies revealed that Cdc45-bound RPA complexed with ssDNA in the 8-10 nucleotide binding mode, but dissociated when RPA covered a 30-mer. Real-time analysis of RPA-ssDNA binding demonstrated that Cdc45 catalytically loaded RPA onto ssDNA. This placement reaction required physical contacts of Cdc45 with the RPA70A subdomain. Our results imply that Cdc45 controlled stabilization of the 8-nt RPA binding mode, the subsequent RPA transition into 30-mer mode and facilitated an ordered binding to ssDNA. We propose that a Cdc45-mediated loading guarantees a seamless deposition of RPA on newly emerging ssDNA at the nascent replication fork.
authors.affiliation: Research Group Biochemistry, Leibniz Institute on Aging-Fritz Lipmann Institute, Beutenbergstrasse 11, D-07745 Jena, Germany.
authors.forename: Anna
authors.initials: A
authors.lastname: Szambowska
authors.affiliation: Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, Josef Schneider Strasse 2, D-97080 Würzburg, Germany.
authors.forename: Ingrid
authors.initials: I
authors.lastname: Tessmer
authors.affiliation: Biocenter Oulu, P.O. Box 5000, 90014 University of Oulu, Finland.
authors.forename: Piotr
authors.initials: P
authors.lastname: Prus
authors.affiliation: Research Group Biochemistry, Leibniz Institute on Aging-Fritz Lipmann Institute, Beutenbergstrasse 11, D-07745 Jena, Germany.|Proteomics Core Facility, Leibniz Institute on Aging-Fritz Lipmann Institute, Beutenbergstrasse 11, D-07745 Jena, Germany.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Schlott
authors.affiliation: Research Group Biochemistry, Leibniz Institute on Aging-Fritz Lipmann Institute, Beutenbergstrasse 11, D-07745 Jena, Germany.|Faculty of Biochemistry and Molecular Medicine, P.O. Box 5000, 90014 University of Oulu, Finland.
authors.forename: Helmut
authors.initials: H
authors.lastname: Pospiech
authors.affiliation: Research Group Biochemistry, Leibniz Institute on Aging-Fritz Lipmann Institute, Beutenbergstrasse 11, D-07745 Jena, Germany.|Center for Molecular Biomedicine, Friedrich-Schiller University, Biochemistry Department, Jena, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Grosse
mesh_terms: D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D011485:Protein Binding; D051716:Replication Protein A / Q000737:chemistry / Q000378:metabolism*
chemical_list: C113571:CDC45 protein, human; D018797:Cell Cycle Proteins; D004277:DNA, Single-Stranded; D051716:Replication Protein A