… as seen in the Rijksmuseum, Amsterdam.
… as seen in the Rijksmuseum, Amsterdam.
J. Hohlbein, L. Aigrain, T.D. Craggs, O. Bermek, O. Potapova, P. Shoolizadeh, N.D.F. Grindley, C.M. Joyce, A.N. Kapanidis, Nature Communications, 4, 2131, 2013, [link], open access
The fidelity of DNA polymerases depends on conformational changes that promote the rejection of incorrect nucleotides before phosphoryl transfer. Here, we combine single-molecule FRET with the use of DNA polymerase I and various fidelity mutants to highlight mechanisms by which active-site side chains influence the conformational transitions and free-energy landscape that underlie fidelity decisions in DNA synthesis. Ternary complexes of high fidelity derivatives with complementary dNTPs adopt mainly a fully closed conformation, whereas a conformation with a FRET value between those of open and closed is sparsely populated. This intermediate-FRET state, which we attribute to a partially closed conformation, is also predominant in ternary complexes with incorrect nucleotides and, strikingly, in most ternary complexes of low-fidelity derivatives for both correct and incorrect nucleotides. The mutator phenotype of the low-fidelity derivatives correlates well with reduced affinity for complementary dNTPs and highlights the partially closed conformation as a primary checkpoint for nucleotide selection.
…who joins the group to pursue his PhD thesis. He is going to study DNA polymerases interacting with DNA by employing single-molecule FRET techniques. He graduated in Molecular Life Sciences here at Wageningen University.
…who join the group for their Bachelor thesis. In close cooperation with the Laboratory of Biochemistry, we are going to look into Arabidopsis lines that express transcriptional regulators (Stef van der Krieken, project with Prof. Dr. Dolf Weijers) and plasma membrane steroid receptors (Stefan Hutten, project with Prof. Dr. Sacco de Vries).
A big welcome to Shazia Farouq (PhD student in the van Amerongen group)! Shazia will take over some of Andy’s projects, who left the group to pursue new endeavours. Thanks to Andy’s work in the last three months, we are now able to detect single fluorescent molecules on our microscope. Hooray!
Picture: Doubly labelled DNA (dyes: Cy3B and ATTO647N) immobilised on a glass surface. The green detection channel is on the left, red detection channel (FRET channel) on the right.
After finishing very late yesterday evening, we proudly present our first image taken with the new setup. Nothing very interesting, just some aggregated, fluorescent latex-beads on a cover slip surface imaged into blue, green and red detection channels of an emCCD camera (after excitation with a green laser). A big thank you goes to John for the laser control software!
Now its time to prepare some buffers, get the oxygen scavenger system working, and image some DNA FRET standards.
After a short flight from Heathrow to Schiphol and some trains and buses later, Andy and I arrived in Wageningen with all our luggage and cardboard-wrapped bikes. On Monday, we had fun opening the boxes from Thorlabs with parts for our new multi-colour TIRF microscope. Opinions on how soon we will be able to detect single, fluorescent molecules seem to differ quite a bit and a couple of bets have been placed, but we accept the challenge and are hoping to share some images in a few weeks time (and collect our winnings).
The picture shows important characteristics of a state-of-the-art microscope: multicolour-excitation path with alternating laser excitation (blue, green, and red lasers), a microscope stage with objective focusing the laser light into a small sample volume, the fluorescence from the sample volume is detected through the same objective, and the light is focussed onto a camera. For more information, click [here].
L. Le Reste, J. Hohlbein, K. Gryte, A.N. Kapanidis, Biophysical Journal, 102, 11, 2658-2668, 2012, [link]
Dark quenchers are chromophores that primarily relax from the excited state to the ground state nonradiatively (i.e., are dark). As a result, they can serve as acceptors for Förster resonance energy transfer experiments without contributing significantly to background in the donor-emission channel, even at high concentrations. Although the advantages of dark quenchers have been exploited for ensemble bioassays, no systematic single-molecule study of dark quenchers has been performed, and little is known about their photophysical properties. Here, we present the first systematic single-molecule study of dark quenchers in conjunction with fluorophores and demonstrate the use of dark quenchers for monitoring multiple interactions and distances in multichromophore systems. Specifically, using double-stranded DNA standards labeled with two fluorophores and a dark quencher (either QSY7 or QSY21), we show that the proximity of a fluorophore and dark quencher can be monitored using the stoichiometry ratio available from alternating laser excitation spectroscopy experiments, either for single molecules diffusing in solution (using a confocal fluorescence) or immobilized on surfaces (using total-internal-reflection fluorescence). The latter experiments allowed characterization of the dark-quencher photophysical properties at the single-molecule level. We also use dark-quenchers to study the affinity and kinetics of binding of DNA Polymerase I (Klenow fragment) to DNA. The measured properties are in excellent agreement with the results of ensemble assays, validating the use of dark quenchers. Because dark-quencher-labeled biomolecules can be used in total-internal-reflection fluorescence experiments at concentrations of 1 μM or more without introducing a significant background, the use of dark quenchers should permit single-molecule Förster resonance energy transfer measurements for the large number of biomolecules that participate in interactions of moderate-to-low affinity.