Noise in the Machine: Alternative Pathway Sampling is the Rule During DNA Replication

Scherr, Matthias J; Safaric, Barbara; Duderstadt, Karen G.

doi:10.1002/bies.201700159

Cited by 15 publications

(15 citation statements)

References 128 publications

(296 reference statements)

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“…Our observations and previous studies (Douglas et al, 2018; Langston et al, 2014; Lewis et al, 2017b) suggest that Pol ε is a stable component of the replisome. However, recent single-molecule experiments have demonstrated that stable components of multi-protein complexes can undergo dynamic exchange when challenged with competing binding partners (Delalez et al, 2010; Graham et al, 2011; Li et al, 2019; Scherr et al, 2018). In particular, complexes held together by multiple weak interactions support high stability in the absence of competing factors but rapid exchange in the presence thereof (van Oijen and Dixon, 2015).…”

Section: Resultsmentioning

confidence: 99%

Tunability of DNA Polymerase Stability during Eukaryotic DNA Replication

et al. 2020

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Structural and biochemical studies have revealed the basic principles of how the replisome duplicates genomic DNA, but little is known about its dynamics during DNA replication. We reconstitute the 34 proteins needed to form the S. cerevisiae replisome and show how changing local concentrations of the key DNA polymerases tunes the ability of the complex to efficiently recycle these proteins or to dynamically exchange them. Particularly, we demonstrate redundancy of the Pol α-primase DNA polymerase activity in replication and show that Pol α-primase and the lagging-strand Pol δ can be re-used within the replisome to support the synthesis of large numbers of Okazaki fragments. This unexpected malleability of the replisome might allow it to deal with barriers and resource challenges during replication of large genomes.

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Section: Resultsmentioning

confidence: 99%

Tunability of DNA Polymerase Stability during Eukaryotic DNA Replication

et al. 2020

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“…Instead a physical encounter is likely required to convert transient breaks into irreversible breaks 30 that are ultimately exposed to the cell during failed repair attempts 31 . The improved throughput of FMT allows for direct characterization of the dynamics of encounters of complex machineries 32 , which were previously out of reach.…”

Section: Discussionmentioning

confidence: 99%

Multiplex flow magnetic tweezers reveal rare enzymatic events with single molecule precision

Agarwal

Duderstadt

2020

Nat Commun

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The application of forces and torques on the single molecule level has transformed our understanding of the dynamic properties of biomolecules, but rare intermediates have remained difficult to characterize due to limited throughput. Here, we describe a method that provides a 100-fold improvement in the throughput of force spectroscopy measurements with topological control, which enables routine imaging of 50,000 single molecules and a 100 million reaction cycles in parallel. This improvement enables detection of rare events in the life cycle of the cell. As a demonstration, we characterize the supercoiling dynamics and drug-induced DNA break intermediates of topoisomerases. To rapidly quantify distinct classes of dynamic behaviors and rare events, we developed a software platform with an automated feature classification pipeline. The method and software can be readily adapted for studies of a broad range of complex, multistep enzymatic pathways in which rare intermediates have escaped classification due to limited throughput.

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“…Recent single-molecule studies have revealed the dynamic behavior of proteins involved in DNA replication. This has changed our view of the replisome from a neat and deterministic machine, to a more "messy" and stochastic system of proteins (Åberg et al 2016;Geertsema and van Oijen 2013;Scherr et al 2018;Xu and Dixon 2018). Proteins bind to the replisome through a plurality of weak interactions.…”

Section: Discussionmentioning

confidence: 99%

When proteins play tag: the dynamic nature of the replisome

2019

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DNA replication, or the copying of DNA, is a fundamental process to all life. The system of proteins that carries out replication, the replisome, encounters many roadblocks on its way. An inability of the replisome to properly overcome these roadblocks will negatively affect genomic integrity which in turn can lead to disease. Over the past decades, efforts by many researchers using a broad array of approaches have revealed roles for many different proteins during the initial response of the replisome upon encountering roadblocks. Here, we revisit what is known about DNA replication and the effect of roadblocks during DNA replication across different organisms. We also address how advances in single-molecule techniques have changed our view of the replisome from a highly stable machine with behavior dictated by deterministic principles to a dynamic system that is controlled by stochastic processes. We propose that these dynamics will play crucial roles in roadblock bypass. Further singlemolecule studies of this bypass will, therefore, be essential to facilitate the in-depth investigation of multi-protein complexes that is necessary to understand complicated collisions on the DNA.

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Noise in the Machine: Alternative Pathway Sampling is the Rule During DNA Replication

Cited by 15 publications

References 128 publications

Tunability of DNA Polymerase Stability during Eukaryotic DNA Replication

Tunability of DNA Polymerase Stability during Eukaryotic DNA Replication

Multiplex flow magnetic tweezers reveal rare enzymatic events with single molecule precision

When proteins play tag: the dynamic nature of the replisome

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