News: The miCube moved to Github

Our open (single-molecule) microscopy project, the #miCube, is now on Github [link]!  The page shows a detailed and updated overview of the components, some information on phasor-based SMLM, and many links to similar open hardware projects.

 

Posted in miCube, news

Pre-print: DNA polymerase β fingers movement revealed by single-molecule FRET suggests a partially closed conformation as a fidelity checkpoint

C. Fijen, M. Mahmoud, R. Kaup, J. Towle-Weicksel, J. Sweasy, and J. Hohlbein, bioRxiv, 2018 [link]

The eukaryotic DNA polymerase β 2018_PolB_overviewplays an important role in cellular DNA repair as it fills gaps in single nucleotide gapped DNA that result from removal of damaged bases. Since defects in DNA repair may lead to cancer and genetic instabilities, Pol β has been extensively studied, especially substrate binding and a fidelity-related conformational change called fingers closing. Here, we applied single-molecule Förster resonance energy transfer to study the conformational dynamics of Pol β. Using an acceptor labelled polymerase and a donor labelled DNA substrate, we measured distance changes associated with DNA binding and fingers movement. Our findings suggest that Pol β does not bend its gapped DNA substrate to the extent related crystal structures indicate: instead, bending seems to be significantly less profound. Furthermore, we visualized dynamic fingers closing in single Pol β-DNA complexes upon addition of complementary nucleotides and derived rates of conformational changes. Additionally, we provide evidence that the fingers close only partially when an incorrect nucleotide is bound. This ajar conformation found in Pol β, a polymerase of the X-family, suggests the existence of an additional fidelity checkpoint similar to what has been previously proposed for a member of the A-family, the bacterial DNA polymerase I.

Posted in open access, pre-print

News: Welcome to Meike Kronenberg and farewell to Carel Fijen

Meike joined the group for her BSc thesis and will work on DNA polymerase beta. Carel successfully defended his PhD thesis in March and decided to continue working on DNA polymerase beta by joining the Sweasy lab at Yale as a postdoctoral research assistent. All the best and we hope receiving new stocks soon!

Posted in news

News: Welcome to Andreas Hentrich and Hans Dekker…

…who joined the group for an 6 week internship (Andreas) and a short capita selecta (Hans). Andreas will try unifying some fragmented software packages for in vivo single particle tracking. Hans will push the limits of our ultrafast pSMLM algorithm for super-resolution microscopy.

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Pre-print: Substrate conformational dynamics drive structure-specific recognition of gapped DNA by DNA polymerase

T.D. Craggs, M. Sustarsic, A. Plochowietz, M. Mosayebi, H. Kaju, A. Cuthbert, J. Hohlbein, L. Domicevica, P.C. Biggin, J.P. K. Doye, A.N. Kapanidis, bioRxiv, 2018 [link]

DNA-binding proteins utilise 2018_CraggsBioRxivPic1.pngdifferent recognition mechanisms to locate their DNA targets. Some proteins recognise specific nucleotide sequences, while many DNA repair proteins interact with specific (often bent) DNA structures. While sequence-specific DNA binding mechanisms have been studied extensively, structure-specific mechanisms remain unclear. Here, we study structure-specific DNA recognition by examining the structure and dynamics of DNA polymerase I (Pol) substrates both alone and in Pol-DNA complexes. Using a rigid-body docking approach based on a network of 73 distance restraints collected using single-molecule FRET, we determined a novel solution structure of the singlenucleotide-gapped DNA-Pol binary complex. The structure was highly consistent with previous crystal structures with regards to the downstream primer-template DNA substrate; further, our structure showed a previously unobserved sharp bend (~120°) in the DNA substrate; we also showed that this pronounced bending of the substrate is present in living bacteria. All-atom molecular dynamics simulations and single-molecule quenching assays revealed that 4-5 nt of downstream gap-proximal DNA are unwound in the binary complex. Coarse-grained simulations on free gapped substrates reproduced our experimental FRET values with remarkable accuracy ( = -0.0025 across 34 independent distances) and revealed that the one-nucleotide-gapped DNA frequently adopted highly bent conformations similar to those in the Pol-bound state (ΔG 7 kT) or duplex (>> 10 kT) DNA. Our results suggest a mechanism by which Pol and other structure-specific DNA-binding proteins locate their DNA targets through sensing of the conformational dynamics of DNA substrates.

Posted in open access, pre-print

Accepted: Precision and accuracy of single-molecule FRET measurements – a worldwide benchmark study

B. Hellenkamp, S. Schmid, O. Doroshenko, O. Opanasyuk, R. Kühnemuth, S. Rezaei Adariani, A. Barth, V. Birkedal, M.E. Bowen, H. Chen, T. Cordes, T. Eilert, C. Fijen, M. Götz, G. Gouridis, E. Gratton, T. Ha, C.A. Hanke, A. Hartmann, J. Hendrix, L.L. Hildebrandt, J. Hohlbein, C.G. Hübner, E. Kallis, A.N. Kapanidis, J.-Y. Kim, G. Krainer, D.C. Lamb, N.K. Lee, E.A. Lemke, B. Levesque, M. Levitus, J.J. McCann, N. Naredi-Rainer, D. Nettels, T. Ngo, R. Qiu, C. Röcker, H. Sanabria, M. Schlierf, B. Schuler, H. Seidel, L. Streit, P. Tinnefeld, S. Tyagi, N. Vandenberk, K.R. Weninger, B. Wünsch, I.S. Yanez-Orozco, J. Michaelis, C.A.M. Seidel, T.D. Craggs, T. Hugel, arXiv, 2017, [link], accepted in Nature Methods

Single-molecule Förster resonance HellenkampArXiv_fig energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards ensuring both the reproducibility and accuracy of measuring FRET efficiencies are currently lacking. Here we report the results of a worldwide, comparative, blind study, in which 20 labs determined the FRET efficiencies of several dye-labeled DNA duplexes. Using a unified and straightforward method, we show that FRET efficiencies can be obtained with a standard deviation between ΔE = +-0.02 and +-0.05. We further suggest an experimental and computational procedure for converting FRET efficiencies into accurate distances. We discuss potential uncertainties in the experiment and the modelling. Our extensive quantitative assessment of intensity-based smFRET measurements and correction procedures serve as an essential step towards validation of distance networks with the ultimate aim to archive reliable structural models of biomolecular systems obtained by smFRET-based hybrid methods.

Posted in accepted, open access, pre-print

News: TA proposal awarded

Good news! The proposal “LocalBioFood: Localisation of bio-macromolecules in food matrices” submitted by Ilja Voets (TU/e) and our lab has been granted by the NWO innovation fund for chemistry [link].

Within the project, the three appointed PhD students will image different components in food, like proteins and carbohydrates, on a sub 100nm length scale using advanced fluorescence microscopy. The project is a collaboration between Eindhoven University of Technology, Wageningen University, Confocal.nl, Unilever and DSM.

Posted in news