Mobile origin-licensing factors confer resistance to conflicts with RNA polymerase

Scherr, Matthias J; Wahab, Syafiq Abd; Remus, Dirk; Duderstadt, Karen G.

doi:10.1016/j.celrep.2022.110531

Cited by 22 publications

(25 citation statements)

References 91 publications

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“…While the cryo-EM grid conditions may alter ORC-nucleosome connections, some of the 2D averages did show their proximity indicative of direct interaction. Another single-molecule study showed that, interestingly, RNA polymerase can push ORC and MCM DH to other locations on DNA, even along with a nucleosome, possibly setting the stage for origins to be placed at new positions 51 .…”

Section: Discussionmentioning

confidence: 99%

Nucleosome-directed replication origin licensing independent of a consensus DNA sequence

Wasserman

Yurieva

et al. 2022

Nat Commun

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The numerous enzymes and cofactors involved in eukaryotic DNA replication are conserved from yeast to human, and the budding yeast Saccharomyces cerevisiae (S.c.) has been a useful model organism for these studies. However, there is a gap in our knowledge of why replication origins in higher eukaryotes do not use a consensus DNA sequence as found in S.c. Using in vitro reconstitution and single-molecule visualization, we show here that S.c. origin recognition complex (ORC) stably binds nucleosomes and that ORC-nucleosome complexes have the intrinsic ability to load the replicative helicase MCM double hexamers onto adjacent nucleosome-free DNA regardless of sequence. Furthermore, we find that Xenopus laevis nucleosomes can substitute for yeast ones in engaging with ORC. Combined with re-analyses of genome-wide ORC binding data, our results lead us to propose that the yeast origin recognition machinery contains the cryptic capacity to bind nucleosomes near a nucleosome-free region and license origins, and that this nucleosome-directed origin licensing paradigm generalizes to all eukaryotes.

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Section: Discussionmentioning

confidence: 99%

Nucleosome-directed replication origin licensing independent of a consensus DNA sequence

Wasserman

Yurieva

et al. 2022

Nat Commun

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“…To demonstrate the range of analysis tasks that can be performed with Mars, we have developed three workflows based on real-world applications. In the first workflow, we determine the rate of transcription of single RNA polymerases by tracking their position as a function of time on long DNAs based on Scherr et al, 2022 . In the second workflow, we demonstrate how to accurately analyze images containing observations of single-molecule FRET based on Hellenkamp et al, 2018 and dynamic FRET arising from conformational switching of Holiday junctions ( Hyeon et al, 2012 ).…”

Section: Resultsmentioning

confidence: 99%

“…Examples of such studies include kinetic studies of DNA replication ( Dequeker et al, 2022 ; Ha et al, 2002 ; Lewis et al, 2020 ), studies of the polymerization of structural elements like actin ( Amann and Pollard, 2001 ), the direct observation of flagellar motor rotation ( Sowa et al, 2005 ), as well as tracking of processes in vivo ( Vizcay-Barrena et al, 2011 ). To illustrate the use of Mars for the analysis of such datasets, this example shows a typical Mars workflow for smTIRF studies of the kinetics of a fluorescently-labeled RNA polymerase transcribing on an immobilized, promoter-containing, 21 kb DNA molecule ( Scherr et al, 2022 ; Figure 3A ). In the presence of all four nucleotides, RNA polymerase could initiate transcription from the promoter and progress on the DNA which could be temporally and spatially visualized by measuring fluorescent emission upon excitation.…”

Section: Resultsmentioning

confidence: 99%

“…Specific details about protein purification and labeling, the microscope set-up, sample preparation, and the imaging procedure can be found in the publication by Scherr et al, 2022 . The raw data accompanying this example workflow is freely available through the Mars tutorials GitHub repository .…”

Section: Methodsmentioning

confidence: 99%

“…Multicolor fluorescence imaging has revealed the conformational dynamics of membrane transport ( Akyuz et al, 2013 ; Erkens et al, 2013 ), molecular states underlying assembly and transcription by RNA polymerase ( Baek et al, 2021 ; Duchi et al, 2016 ), and DNA replication dynamics ( Duderstadt et al, 2016 ; Scherr et al, 2018 ; Ticau et al, 2015 ). These approaches have been combined with spatial tracking on biological structures to clarify how exchange events and conformational changes modulate the function of motor proteins, as well as replication, transcription, and DNA repair machineries ( Crickard et al, 2020 ; Lewis et al, 2020 ; Niekamp et al, 2021 ; Scherr et al, 2022 ). Biological macromolecules are routinely attached to microspheres which allow for controlled studies of the forces and torques involved in basic biological reactions ( Agarwal and Duderstadt, 2020 ; Dulin et al, 2013 ; Neuman and Nagy, 2008 ; Revyakin et al, 2006 ).…”

Section: Introductionmentioning

confidence: 99%

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Mars, a molecule archive suite for reproducible analysis and reporting of single-molecule properties from bioimages

Huisjes

Retzer

Scherr

et al. 2022

eLife

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The rapid development of new imaging approaches is generating larger and more complex datasets, revealing the time evolution of individual cells and biomolecules. Single-molecule techniques, in particular, provide access to rare intermediates in complex, multistage molecular pathways. However, few standards exist for processing these information-rich datasets, posing challenges for wider dissemination. Here, we present Mars, an open-source platform for storing and processing image-derived properties of biomolecules. Mars provides Fiji/ImageJ2 commands written in Java for common single-molecule analysis tasks using a Molecule Archive architecture that is easily adapted to complex, multistep analysis workflows. Three diverse workflows involving molecule tracking, multichannel fluorescence imaging, and force spectroscopy, demonstrate the range of analysis applications. A comprehensive graphical user interface written in JavaFX enhances biomolecule feature exploration by providing charting, tagging, region highlighting, scriptable dashboards, and interactive image views. The interoperability of ImageJ2 ensures Molecule Archives can easily be opened in multiple environments, including those written in Python using PyImageJ, for interactive scripting and visualization. Mars provides a flexible solution for reproducible analysis of image-derived properties, facilitating the discovery and quantitative classification of new biological phenomena with an open data format accessible to everyone.

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De novo fabrication of custom-sequence plasmids for the synthesis of long DNA constructs with extrahelical features

Ramírez Montero,

Liu,

Dekker

2024

Biophysical Journal

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Mobile origin-licensing factors confer resistance to conflicts with RNA polymerase

Cited by 22 publications

References 91 publications

Nucleosome-directed replication origin licensing independent of a consensus DNA sequence

Nucleosome-directed replication origin licensing independent of a consensus DNA sequence

Mars, a molecule archive suite for reproducible analysis and reporting of single-molecule properties from bioimages

De novo fabrication of custom-sequence plasmids for the synthesis of long DNA constructs with extrahelical features

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