Using single-molecule FRET to probe the nucleotide-dependent conformational landscape of polymerase b-DNA complexes

Carel Fijen, Mariam M. Mahmoud, Meike Kronenberg, Rebecca Kaup, Mattia Fontana, Jamie B. Towle-Weicksel, Joann B. Sweasy, Johannes Hohlbein

Research output: Contribution to journalArticlepeer-review

3 Scopus citations


Eukaryotic DNA polymerase b (Pol b) 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 b 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 b. First, using a doubly labeled DNA construct, we show that Pol b bends the gapped DNA substrate less than indicated by previously reported crystal structures. Second, using acceptor-labeled Pol b and donor-labeled DNA, we visualized dynamic fingers closing in single Pol b-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 b, 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 b reported here provide new insights into the delicate mechanisms of prechemistry nucleotide selection.

Original languageEnglish (US)
Pages (from-to)9012-9020
Number of pages9
JournalJournal of Biological Chemistry
Issue number27
StatePublished - Jul 3 2020

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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