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Mechanism of assembly of an elongation-competent SARS-CoV-2 replication transcription complex

Klein, M. (author), Bera, Subhas C. (author), Anderson, Thomas K. (author), Wang, Bing (author), Papini, Flavia S. (author), Arnold, Jamie J. (author), Cameron, Craig E. (author), Depken, S.M. (author), Dulin, D. (author)

journal article 2023

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A kinetic model predicts SpCas9 activity, improves off-target classification, and reveals the physical basis of targeting fidelity

Eslami Mossallam, B. (author), Klein, M. (author), Smagt, Constantijn V.D. (author), Sanden, Koen V.D. (author), Jones, Stephen K. (author), Hawkins, John A. (author), Finkelstein, Ilya J. (author), Depken, S.M. (author)

The S. pyogenes (Sp) Cas9 endonuclease is an important gene-editing tool. SpCas9 is directed to target sites based on complementarity to a complexed single-guide RNA (sgRNA). However, SpCas9-sgRNA also binds and cleaves genomic off-targets with only partial complementarity. To date, we lack the ability to predict cleavage and binding activity...

journal article 2022

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Quantitative parameters of bacterial RNA polymerase open-complex formation, stabilization and disruption on a consensus promoter

Bera, Subhas C. (author), America, Pim P.B. (author), Maatsola, Santeri (author), Seifert, Mona (author), Ostrofet, Eugeniu (author), Cnossen, J.P. (author), Spermann, Monika (author), Depken, S.M. (author), Dulin, D. (author)

Transcription initiation is the first step in gene expression, and is therefore strongly regulated in all domains of life. The RNA polymerase (RNAP) first associates with the initiation factor σ to form a holoenzyme, which binds, bends and opens the promoter in a succession of reversible states. These states are critical for transcription...

journal article 2022

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High-throughput single-molecule experiments reveal heterogeneity, state switching, and three interconnected pause states in transcription

Janissen, R. (author), Eslami Mossallam, B. (author), Artsimovitch, Irina (author), Depken, S.M. (author), Dekker, N.H. (author)

Pausing by bacterial RNA polymerase (RNAp) is vital in the recruitment of regulatory factors, RNA folding, and coupled translation. While backtracking and intra-structural isomerization have been proposed to trigger pausing, our mechanistic understanding of backtrack-associated pauses and catalytic recovery remains incomplete. Using high...

journal article 2022

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Systematic decomposition of sequence determinants governing CRISPR/Cas9 specificity

Fu, Rongjie (author), He, Wei (author), Dou, Jinzhuang (author), Villarreal, Oscar D. (author), Bedford, Ella (author), Wang, Helen (author), Hou, Connie (author), Zhang, Liang (author), Depken, S.M. (author)

The specificity of CRISPR/Cas9 genome editing is largely determined by the sequences of guide RNA (gRNA) and the targeted DNA, yet the sequence-dependent rules underlying off-target effects are not fully understood. To systematically explore the sequence determinants governing CRISPR/Cas9 specificity, here we describe a dual-target system to...

journal article 2022

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The nucleotide addition cycle of the SARS-CoV-2 polymerase

Bera, Subhas Chandra (author), Seifert, Mona (author), Kirchdoerfer, Robert N. (author), van Nies, Pauline (author), Wubulikasimu, Yibulayin (author), Quack, Salina (author), Papini, Flávia S. (author), Depken, S.M. (author), Dulin, D. (author)

Coronaviruses have evolved elaborate multisubunit machines to replicate and transcribe their genomes. Central to these machines are the RNA-dependent RNA polymerase subunit (nsp12) and its intimately associated cofactors (nsp7 and nsp8). We use a high-throughput magnetic-tweezers approach to develop a mechanochemical description of this core...

journal article 2021

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Inhibition of SARS-CoV-2 polymerase by nucleotide analogs from a single-molecule perspective

Seifert, Mona (author), Bera, Subhas C. (author), van Nies, Pauline (author), Kirchdoerfer, Robert N. (author), Shannon, Ashleigh (author), Le, Thi Tuyet Nhung (author), Meng, Xiangzhi (author), Depken, S.M. (author), Dulin, David (author)

The absence of 'shovel-ready' anti-coronavirus drugs during vaccine development has exceedingly worsened the SARS-CoV-2 pandemic. Furthermore, new vaccine-resistant variants and coronavirus outbreaks may occur in the near future, and we must be ready to face this possibility. However, efficient antiviral drugs are still lacking to this day,...

journal article 2021

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Temperature controlled high-throughput magnetic tweezers show striking difference in activation energies of replicating viral RNA-dependent RNA polymerases

Seifert, Mona (author), van Nies, Pauline (author), Papini, Flávia S. (author), Arnold, Jamie J. (author), Poranen, Minna M. (author), Cameron, Craig E. (author), Depken, S.M. (author), Dulin, D. (author)

RNA virus survival depends on efficient viral genome replication, which is performed by the viral RNA dependent RNA polymerase (RdRp). The recent development of high throughput magnetic tweezers has enabled the simultaneous observation of dozens of viral RdRp elongation traces on kilobases long templates, and this has shown that RdRp...

journal article 2020

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Argonaute bypasses cellular obstacles without hindrance during target search

Cui, Thijs (author), Klein, M. (author), Hegge, Jorrit W. (author), Chandradoss, S.D. (author), van der Oost, John (author), Depken, S.M. (author), Joo, C. (author)

Argonaute (Ago) proteins are key players in both gene regulation (eukaryotes) and host defense (prokaryotes). Acting on single-stranded nucleic-acid substrates, Ago relies on base pairing between a small nucleic-acid guide and its complementary target sequences for specificity. To efficiently scan nucleic-acid chains for targets, Ago diffuses...

journal article 2019

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Pausing controls branching between productive and non-productive pathways during initial transcription in bacteria

Dulin, D. (author), Bauer, David L.V. (author), Malinen, Anssi M. (author), Bakermans, Jacob J.W. (author), Kaller, Martin (author), Morichaud, Zakia (author), Petushkov, Ivan (author), Depken, S.M. (author), Brodolin, Konstantin (author), Kulbachinskiy, Andrey (author), Kapanidis, Achillefs N. (author)

Transcription in bacteria is controlled by multiple molecular mechanisms that precisely regulate gene expression. It has been recently shown that initial RNA synthesis by the bacterial RNA polymerase (RNAP) is interrupted by pauses; however, the pausing determinants and the relationship of pausing with productive and abortive RNA synthesis...

journal article 2018

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Kinetic analysis methods applied to single motor protein trajectories

Nord, A. L. (author), Pols, AAJ (author), Depken, S.M. (author), Pedaci, F. (author)

Molecular motors convert chemical or electrical energy into mechanical displacement, either linear or rotary. Under ideal circumstances, single-molecule measurements can spatially and temporally resolve individual steps of the motor, revealing important properties of the underlying mechanochemical process. Unfortunately, steps are often hard...

journal article 2018

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Hybridization Kinetics Explains CRISPR-Cas Off-Targeting Rules

Klein, M. (author), Eslami Mossallam, B. (author), Gonzalez Arroyo, D.W. (author), Depken, S.M. (author)

Due to their specificity, efficiency, and ease of programming, CRISPR-associated nucleases are popular tools for genome editing. On the genomic scale, these nucleases still show considerable off-target activity though, posing a serious obstacle to the development of therapies. Off targeting is often minimized by choosing especially high...

journal article 2018

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What is all this fuss about Tus?: Comparison of recent findings from biophysical and biochemical experiments

Berghuis, B.A. (author), Raducanu, Vlad-Stefan (author), Elshenawy, Mohamed M. (author), Jergic, Slobodan (author), Depken, S.M. (author), Dixon, NE (author), Hamdan, Samir M. (author), Dekker, N.H. (author)

Synchronizing the convergence of the two-oppositely moving DNA replication machineries at specific termination sites is a tightly coordinated process in bacteria. In Escherichia coli, a “replication fork trap” – found within a chromosomal region where forks are allowed to enter but not leave – is set by the protein–DNA roadblock Tus–Ter. The...

journal article 2017

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Why Argonaute is needed to make microRNA target search fast and reliable

Klein, M. (author), Chandradoss, S.D. (author), Depken, S.M. (author), Joo, C. (author)

MicroRNA (miRNA) interferes with the translation of cognate messenger RNA (mRNA) by finding, preferentially binding, and marking it for degradation. To facilitate the search process, Argonaute (Ago) proteins come together with miRNA, forming a dynamic search complex. In this review we use the language of free-energy landscapes to discuss...

journal article 2017

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Crowding-induced transcriptional bursts dictate polymerase and nucleosome density profiles along genes

van den Berg, A.A. (author), Depken, S.M. (author)

During eukaryotic transcription, RNA polymerase (RNAP) translocates along DNA molecules covered with nucleosomes and other DNA binding proteins. Though the interactions between a single nucleosome and RNAP are by now fairly well understood, this understanding has not been synthesized into a description of transcription on crowded genes, where...

journal article 2017

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Signatures of Nucleotide Analog Incorporation by an RNA-Dependent RNA Polymerase Revealed Using High-Throughput Magnetic Tweezers

Dulin, D. (author), Arnold, Jamie J. (author), van Laar, T. (author), Oh, Hyung Suk (author), Lee, Cheri (author), Perkins, Angela L. (author), Harki, Daniel A. (author), Depken, S.M. (author), Cameron, Craig E. (author), Dekker, N.H. (author)

RNA viruses pose a threat to public health that is exacerbated by the dearth of antiviral therapeutics. The RNA-dependent RNA polymerase (RdRp) holds promise as a broad-spectrum, therapeutic target because of the conserved nature of the nucleotide-substrate-binding and catalytic sites. Conventional, quantitative, kinetic analysis of antiviral...

journal article 2017

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Cas3-Derived Target DNA Degradation Fragments Fuel Primed CRISPR Adaptation

Künne, Tim (author), Kieper, S.N. (author), Bannenberg, Jasper W. (author), Vogel, Anne I M (author), Miellet, Willem R. (author), Klein, M. (author), Depken, S.M. (author), Suarez-Diez, Maria (author), Brouns, S.J.J. (author)

Prokaryotes use a mechanism called priming to update their CRISPR immunological memory to rapidly counter revisiting, mutated viruses, and plasmids. Here we have determined how new spacers are produced and selected for integration into the CRISPR array during priming. We show that Cas3 couples CRISPR interference to adaptation by producing...

journal article 2016

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Elongation-Competent Pauses Govern the Fidelity of a Viral RNA-Dependent RNA Polymerase

Dulin, D. (author), Vilfan, I.D. (author), Berghuis, B.A. (author), Hage, S. (author), Bamford, D.H. (author), Poranen, M.M. (author), Depken, S.M. (author), Dekker, N.H. (author)

RNA viruses have specific mutation rates that balance the conflicting needs of an evolutionary response to host antiviral defenses and avoidance of the error catastrophe. While most mutations are known to originate in replication errors, difficulties of capturing the underlying dynamics have left the mechanochemical basis of viral mutagenesis...

journal article 2015

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Untangling reaction pathways through modern approaches to high-throughput single-molecule force-spectroscopy experiments

Dulin, D. (author), Berghuis, B.A. (author), Depken, S.M. (author), Dekker, N.H. (author)

Single-molecule experiments provide a unique means for real-time observation of the activity of individual biomolecular machines. Through such techniques, insights into the mechanics of for example, polymerases, helicases, and packaging motors have been gleaned. Here we describe the recent advances in single-molecule force spectroscopy...

journal article 2015

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Strand separation establishes a sustained lock at the Tus-Ter replication fork barrier

Berghuis, B.A. (author), Dulin, D. (author), Xu, Zhi Qiang (author), van Laar, T. (author), Cross, B. (author), Janissen, R. (author), Jergic, Slobodan (author), Dixon, Nicholas E. (author), Depken, S.M. (author), Dekker, N.H. (author)

The bidirectional replication of a circular chromosome by many bacteria necessitates proper termination to avoid the head-on collision of the opposing replisomes. In Escherichia coli, replisome progression beyond the termination site is prevented by Tus proteins bound to asymmetric Ter sites. Structural evidence indicates that strand...

journal article 2015