Published: Substrate conformational dynamics facilitate 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, Nucleic Acid Research, gkz797, 2019, [link]

DNA-binding proteins utilise different recognition mechanisms to locate their DNA targets; some proteins recognise specific DNA sequences, while others interact with specific DNA structures. While sequence-specific DNA binding has been studied extensively, structure-specific recognition mechanisms remain unclear. Here, we study structure-specific DNA recognition by examining the structure and dynamics of DNA polymerase I Klenow Fragment (Pol) substrates both alone and in DNA–Pol complexes. Using a docking approach based on a network of 73 distances collected using single-molecule FRET, we determined a novel solution structure of the single-nucleotide-gapped DNA–Pol binary complex. The structure resembled existing crystal structures with regards to the downstream primer-template DNA substrate, and revealed a previously unobserved sharp bend (∼120°) in the DNA substrate; this pronounced bend was present in living cells. MD simulations and single-molecule assays also revealed that 4–5 nt of downstream gap-proximal DNA are unwound in the binary complex. Further, experiments and coarse-grained modelling showed the substrate alone frequently adopts bent conformations with 1–2 nt fraying around the gap, suggesting a mechanism wherein Pol recognises a pre-bent, partially-melted conformation of gapped DNA. We propose a general mechanism for substrate recognition by structure-specific enzymes driven by protein sensing of the conformational dynamics of their DNA substrates.

 

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Pre-print: Design principles of a minimal auxin response system

H. Kato, S.K. Mutte, H. Suzuki, I.  Crespo, S.  Das, T. Radoeva, M. Fontana, Y. Yoshitake, E. Hainiwa, W. van den Berg, S. Lindhoud, J. Hohlbein, J.W. Borst, D.R. Boer, R. Nishihama, T. Kohchi, D. Weijers, bioRxiv, 2019, [link]

Auxin controls numerous growth processes in land plants through a gene expression system that modulates ARF transcription factor activity. Gene duplications in families encoding auxin response components have generated tremendous complexity in most land plants, and neofunctionalization enabled various unique response outputs during development. However, it is unclear what fundamental biochemical principles underlie this complex response system. By studying the minimal system in Marchantia polymorpha, we derive an intuitive and simple model where a single auxin-dependent A-ARF activates gene expression. It is antagonized by an auxin-independent B-ARF that represses common target genes. Expression patterns of both ARF proteins define developmental zones where auxin response is permitted, quantitatively tuned, or prevented. This fundamental design likely represents the ancestral system, and formed the basis for inflated, complex systems.

ARF-Marchantia

Publication: Visualisation of dCas9 target search in vivo using an open-microscopy framework

K.J.A. Martens, S. van Beljouw, S. van der Els, J.N.A. Vink, S. Baas, G.A. Vogelaar, S.J.J. Brouns, P. van Baarlen, M. Kleerebezem, J. Hohlbein, Nature Communications, 10, 3552, 2019, [link]

CRISPR-Cas9 is widely used in genomic editing, but the kinetics of target search and its relation to the cellular concentration of Cas9 have remained elusive. Effective target search requires constant screening of the protospacer adjacent motif (PAM) and a 30 ms upper limit for screening was recently found. To further quantify the rapid switching between DNA-bound and freely-diffusing states of dCas9, we developed an open-microscopy framework, the miCube, and introduce Monte-Carlo diffusion distribution analysis (MC-DDA). Our analysis reveals that dCas9 is screening PAMs 40% of the time in Gram-positive Lactoccous lactis, averaging 17 ± 4 ms per binding event. Using heterogeneous dCas9 expression, we determine the number of cellular target-containing plasmids and derive the copy number dependent Cas9 cleavage. Furthermore, we show that dCas9 is not irreversibly bound to target sites but can still interfere with plasmid replication. Taken together, our quantitative data facilitates further optimization of the CRISPR-Cas toolbox.

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Pre-print: Direct visualization of native CRISPR target search in live bacteria reveals Cascade DNA surveillance mechanism

J.N.A. Vink, K.J.A. Martens, M. Vlot, R.E. McKenzie, C. Almendros, B. Estrada Bonilla, D.J.W. Brocken, J. Hohlbein, S.J.J. Brouns, bioRxiv, 2019, [link]

CRISPR-Cas systems encode RNA-guided surveillance complexes to find and cleave invading DNA elements. While it is thought that invaders are neutralized minutes after cell entry, the mechanism and kinetics of target search and its impact on CRISPR protection levels have remained unknown. Here we visualized individual Cascade complexes in a native type I CRISPR-Cas system. We uncovered an exponential relationship between Cascade copy number and CRISPR interference levels, pointing to a time-driven arms race between invader replication and target search, in which 20 Cascade complexes provide 50% protection. Driven by PAM-interacting subunit Cas8e, Cascade spends half its search time rapidly probing DNA (∼30 ms) in the nucleoid. We further demonstrate that target DNA transcription and CRISPR arrays affect the integrity of Cascade and impact CRISPR interference. Our work establishes the mechanism of cellular DNA surveillance by Cascade that allows the timely detection of invading DNA in a crowded, DNA-packed environment.

2019_Vink_bioRxiv

Pre-print: An open microscopy framework suited for tracking dCas9 in live bacteria

K.J.A. Martens, S. van Beljouw, S. van der Els, S. Baas, J.N.A. Vink, S.J.J. Brouns, P. van Baarlen, M. Kleerebezem, J. Hohlbein, bioRxiv, 2018 [link]

Super-resolution microscopy is frequently employedin 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.

Published: Precision and accuracy of single-molecule FRET measurements—a multi-laboratory benchmark study

B. Hellenkamp, S. Schmid, O. Doroshenko, O. Opanasyuk, R. Kühnemuth, S. Rezaei Adariani, B. Ambrose, M. Aznauryan, A. Barth, V. Birkedal, M.E. Bowen, H. Chen, T. Cordes, T. Eilert, C. Fijen, C. Gebhardt, M. Götz, G. Gouridis, E. Gratton, T. Ha, P. Hao, C.A. Hanke, A. Hartmann, J. Hendrix, L.L. Hildebrandt, V. Hirschfeld, J. Hohlbein, B.g Hua, 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, N.C. Robb, C. Röcker, H. Sanabria, M. Schlierf, T. Schröder, B. Schuler, H. Seidel, L. Streit, J. Thurn, P. Tinnefeld, S. Tyagi, N. Vandenberk, A. Manuel Vera, K.R. Weninger, B. Wünsch, I.S. Yanez-Orozco, J. Michaelis, C.A.M. Seidel, T.D. Craggs, T. Hugel, Nature Methods, 15, 669, 2018, [link], preprint on arXiv: [link]

Single-molecule Förster resonance energytransfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ± 0.02 and ± 0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods.

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 βplays 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.

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