TY - JOUR
T1 - Using single-molecule FRET to probe the nucleotide-dependent conformational landscape of polymerase b-DNA complexes
AU - Fijen, Carel
AU - Mahmoud, Mariam M.
AU - Kronenberg, Meike
AU - Kaup, Rebecca
AU - Fontana, Mattia
AU - Towle-Weicksel, Jamie B.
AU - Sweasy, Joann B.
AU - Hohlbein, Johannes
N1 - Publisher Copyright:
© 2020 Fijen et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.
PY - 2020/7/3
Y1 - 2020/7/3
N2 - 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.
AB - 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.
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U2 - 10.1074/jbc.ra120.013049
DO - 10.1074/jbc.ra120.013049
M3 - Article
C2 - 32385112
AN - SCOPUS:85087532715
SN - 0021-9258
VL - 295
SP - 9012
EP - 9020
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 27
ER -