University of Oulu

Anna Szambowska, Ingrid Tessmer, Piotr Prus, Bernhard Schlott, Helmut Pospiech, Frank Grosse; Cdc45-induced loading of human RPA onto single-stranded DNA. Nucleic Acids Res 2017; 45 (6): 3217-3230. doi: 10.1093/nar/gkw1364

Cdc45-induced loading of human RPA onto single-stranded DNA

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Author: Szambowska, Anna1; Tessmer, Ingrid2; Prus, Piotr3;
Organizations: 1Research Group Biochemistry, Leibniz Institute on Aging-Fritz Lipmann Institute, Beutenbergstrasse 11, D-07745 Jena, Germany
2Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, Josef Schneider Strasse 2, D-97080 Würzburg, Germany
3Biocenter Oulu, P.O. Box 5000, 90014 University of Oulu, Finland
4Faculty of Biochemistry and Molecular Medicine, P.O. Box 5000, 90014 University of Oulu, Finland
5Center for Molecular Biomedicine, Friedrich-Schiller University, Biochemistry Department, Jena, Germany
Format: article
Version: published version
Access: open
Online Access: PDF Full Text (PDF, 4.2 MB)
Persistent link: http://urn.fi/urn:nbn:fi-fe201707077671
Language: English
Published: Oxford University Press, 2017
Publish Date: 2017-07-07
Description:

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.
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Series: Nucleic acids research
ISSN: 0305-1048
ISSN-E: 1362-4962
ISSN-L: 0305-1048
Volume: 45
Issue: 6
Pages: 3217 - 3230
DOI: 10.1093/nar/gkw1364
OADOI: https://oadoi.org/10.1093/nar/gkw1364
Type of Publication: A1 Journal article – refereed
Field of Science: 1182 Biochemistry, cell and molecular biology
Subjects:
dna
Funding: The Fritz Lipmann Institute (FLI) is member of the Science Association ‘Gottfried Wilhelm Leibniz’ (WGL) and is financially supported by the Federal Government of Germany and the State of Thuringia; Rudolf Virchow Center by the German Research Council (Deutsche Forschungsgemeinschaft, DFG) [FZ 82]. Funding for open access charge: Federal Government of Germany and the State of Thuringia.
Copyright information: © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com
  https://creativecommons.org/licenses/by-nc/4.0/