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Using single-molecule FRET to probe the nucleotide-dependent conformational landscape of polymerase ß-DNA complexes.
Fijen, Carel; Mahmoud, Mariam M; Kronenberg, Meike; Kaup, Rebecca; Fontana, Mattia; Towle-Weicksel, Jamie B; Sweasy, Joann B; Hohlbein, Johannes.
Afiliación
  • Fijen C; Laboratory of Biophysics, Wageningen University & Research, Wageningen, The Netherlands; Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, Connecticut, USA. Electronic address: carelfijen89@gmail.com.
  • Mahmoud MM; Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, Connecticut, USA.
  • Kronenberg M; Laboratory of Biophysics, Wageningen University & Research, Wageningen, The Netherlands.
  • Kaup R; Laboratory of Biophysics, Wageningen University & Research, Wageningen, The Netherlands.
  • Fontana M; Laboratory of Biophysics, Wageningen University & Research, Wageningen, The Netherlands.
  • Towle-Weicksel JB; Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, Connecticut, USA.
  • Sweasy JB; Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, Connecticut, USA.
  • Hohlbein J; Laboratory of Biophysics, Wageningen University & Research, Wageningen, The Netherlands; Microspectroscopy Research Facility, Wageningen University & Research, Wageningen, The Netherlands. Electronic address: johannes.hohlbein@wur.nl.
J Biol Chem ; 295(27): 9012-9020, 2020 07 03.
Article en En | MEDLINE | ID: mdl-32385112
Eukaryotic DNA polymerase ß (Pol ß) plays an important role in cellular DNA repair, as it fills short gaps in dsDNA that result from removal of damaged bases. Since defects in DNA repair may lead to cancer and genetic instabilities, Pol ß has been extensively studied, especially its mechanisms for substrate binding and a fidelity-related conformational change referred to as "fingers closing." Here, we applied single-molecule FRET to measure distance changes associated with DNA binding and prechemistry fingers movement of human Pol ß. First, using a doubly labeled DNA construct, we show that Pol ß bends the gapped DNA substrate less than indicated by previously reported crystal structures. Second, using acceptor-labeled Pol ß and donor-labeled DNA, we visualized dynamic fingers closing in single Pol ß-DNA complexes upon addition of complementary nucleotides and derived rates of conformational changes. We further found that, while incorrect nucleotides are quickly rejected, they nonetheless stabilize the polymerase-DNA complex, suggesting that Pol ß, when bound to a lesion, has a strong commitment to nucleotide incorporation and thus repair. In summary, the observation and quantification of fingers movement in human Pol ß reported here provide new insights into the delicate mechanisms of prechemistry nucleotide selection.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: ADN / ADN Polimerasa beta Límite: Humans Idioma: En Revista: J Biol Chem Año: 2020 Tipo del documento: Article Pais de publicación: Estados Unidos

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: ADN / ADN Polimerasa beta Límite: Humans Idioma: En Revista: J Biol Chem Año: 2020 Tipo del documento: Article Pais de publicación: Estados Unidos