Biology subjects

Hohlbein, J.

Publications and source records attributed to Hohlbein, J..

5 recordsLinked to original sources

bioRxiv2018

An open microscopy framework suited for tracking dCas9 in live bacteria

Super-resolution microscopy is frequently employed in the life sciences, but the number of freely accessible and affordable microscopy frameworks, especially for single particle tracking photo-activation localization microscopy (sptPALM), remains limited. To this end, we designed the miCube: a versatile super-resolution capable fluorescence microscope, which combines high spatiotemporal resolution, good adaptability, low price, and easy installation. By providing all details, we hope to enable interested researchers to build an identical or derivative instrument. The capabilities of the miCube are assessed with a novel sptPALM assay relying on the heterogeneous expression of target genes. Here, we elucidate mechanistic details of catalytically inactive Cas9 (dead Cas9) in live Lactococcus lactis. We show that, lacking specific DNA target sites, the binding and unbinding of dCas9 to DNA can be described using simplified rate constants of kbound[->]free = 30-80 s-1 and kfree[->]bound = 15-40 s-1. Moreover, after providing specific DNA target sites via DNA plasmids, the plasmid-bound dCas9 population size decreases with increasing dCas9 copy number via a mono-exponential decay, indicative of simple disassociation kinetics.

biophysics↗

bioRxiv2018

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

The eukaryotic DNA polymerase {beta} plays 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 {beta} has been extensively studied, especially substrate binding and a fidelity-related conformational change called \"fingers closing\". Here, we applied single-molecule Forster resonance energy transfer to study the conformational dynamics of Pol {beta}. 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 {beta} 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 {beta}-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 {beta}, 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.

biophysics↗

bioRxiv2018

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

DNA-binding proteins utilise different 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{degrees}) 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. Coarsegrained simulations on free gapped substrates reproduced our experimental FRET values with remarkable accuracy (<{Delta}FRET> = -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 ({Delta}G < 4 kT); such conformations were much less accessible to nicked (> 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.\n\nSignificance StatementMost genetic processes, including DNA replication, repair and transcription, rely on DNA-binding proteins locating specific sites on DNA; some sites contain a specific sequence, whereas others present a specific structure. While sequence-specific recognition has a clear physical basis, structure-specific recognition mechanisms remain obscure. Here, we use single-molecule FRET and computer simulations to show that the conformational dynamics of an important repair intermediate (1nt-gapped DNA) act as central recognition signals for structure-specific binding by DNA polymerase I (Pol). Our conclusion is strongly supported by a novel solution structure of the Pol-DNA complex wherein the gapped-DNA is significantly bent. Our iterative approach combining precise single-molecule measurements with molecular modelling is general and can elucidate the structure and dynamics for many large biomachines.

biophysics↗

bioRxiv2017

Simple nanofluidic devices for high-throughput, non-equilibrium studies at the single-molecule level

Single-molecule detection schemes offer powerful means to overcome static and dynamic heterogeneity inherent to complex samples. Probing chemical and biological interactions and reactions with high throughput and time resolution, however, remains challenging and often requires surface-immobilized entities. Here, utilizing camera-based fluorescence microscopy, we present glass-made nanofluidic devices in which fluorescently labelled molecules flow through nanochannels that confine their diffusional movement. The first design features an array of parallel nanochannels for high-throughput analysis of molecular species under equilibrium conditions allowing us to record 200.000 individual localization events in just 10 minutes. Using these localizations for single particle tracking, we were able to obtain accurate flow profiles including flow speeds and diffusion coefficients inside the channels.\n\nA second design featuring a T-shaped nanochannel enables precise mixing of two different species as well as the continuous observation of chemical reactions. We utilized the design to visualize enzymatically driven DNA synthesis in real time and at the single-molecule level. Based on our results, we are convinced that the versatility and performance of the nanofluidic devices will enable numerous applications in the life sciences.

biophysics↗

bioRxiv2017

Phasor based single-molecule localization microscopy in 3D (pSMLM-3D): an algorithm for MHz localization rates using standard CPUs

We present a fast and model-free 2D and 3D single-molecule localization algorithm that allows more than 3 million localizations per second on a standard multi-core CPU with localization accuracies in line with the most accurate algorithms currently available. Our algorithm converts the region of interest around a point spread function (PSF) to two phase vectors (phasors) by calculating the first Fourier coefficients in both x- and y-direction. The angles of these phasors are used to localize the center of the single fluorescent emitter, and the ratio of the magnitudes of the two phasors is a measure for astigmatism, which can be used to obtain depth information (z-direction). Our approach can be used both as a stand-alone algorithm for maximizing localization speed and as a first estimator for more time consuming iterative algorithms.

biophysics↗