scholarly journals Single-Molecule Dynamics at a Bacterial Replication Fork after Nutritional Downshift or Chemically Induced Block in Replication

mSphere ◽  
2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Rogelio Hernández-Tamayo ◽  
Hannah Schmitz ◽  
Peter L. Graumann
Keyword(s):  
Dna Damage ◽  
Bacillus Subtilis ◽  
Single Molecule ◽  
Stringent Response ◽  
Stress Conditions ◽  
High Plasticity ◽  
Replication Forks ◽  
Content Type ◽  
Single Molecule Dynamics ◽  
Molecule Dynamics

ABSTRACT Replication forks must respond to changes in nutrient conditions, especially in bacterial cells. By investigating the single-molecule dynamics of replicative helicase DnaC, DNA primase DnaG, and lagging-strand polymerase DnaE in the model bacterium Bacillus subtilis, we show that proteins react differently to stress conditions in response to transient replication blocks due to DNA damage, to inhibition of the replicative polymerase, or to downshift of serine availability. DnaG appears to be recruited to the forks by a diffusion and capture mechanism, becomes more statically associated after the arrest of polymerase, but binds less frequently after fork blocks due to DNA damage or to nutritional downshift. These results indicate that binding of the alarmone (p)ppGpp due to stringent response prevents DnaG from binding to forks rather than blocking bound primase. Dissimilar behavior of DnaG and DnaE suggests that both proteins are recruited independently to the forks rather than jointly. Turnover of all three proteins was increased during replication block after nutritional downshift, different from the situation due to DNA damage or polymerase inhibition, showing high plasticity of forks in response to different stress conditions. Forks persisted during all stress conditions, apparently ensuring rapid return to replication extension. IMPORTANCE All cells need to adjust DNA replication, which is achieved by a well-orchestrated multiprotein complex, in response to changes in physiological and environmental conditions. For replication forks, it is extremely challenging to meet with conditions where amino acids are rapidly depleted from cells, called the stringent response, to deal with the inhibition of one of the centrally involved proteins or with DNA modifications that arrest the progression of forks. By tracking helicase (DnaC), primase (DnaG), and polymerase (DnaE), central proteins of Bacillus subtilis replication forks, at a single molecule level in real time, we found that interactions of the three proteins with replication forks change in different manners under different stress conditions, revealing an intriguing plasticity of replication forks in dealing with replication obstacles. We have devised a new tool to determine rates of exchange between static movement (binding to a much larger complex) and free diffusion, showing that during stringent response, all proteins have highly increased exchange rates, slowing down overall replication, while inactivation of polymerase or replication roadblocks leaves forks largely intact, allowing rapid restart once obstacles are removed.

scholarly journals Single molecule dynamics at a bacterial replication fork after nutritional downshift

2020 ◽  
Author(s):  
Rogelio Hernández-Tamayo ◽  
Hannah Schmitz ◽  
Peter L. Graumann
Keyword(s):  
Dna Damage ◽  
Single Molecule ◽  
Stringent Response ◽  
Stress Conditions ◽  
High Plasticity ◽  
Bacterial Cells ◽  
Single Molecule Dynamics ◽  
Replication Block ◽  
Bacterial Replication ◽  
Molecule Dynamics

ABSTRACTReplication forks must respond to changes in nutrient conditions, especially in bacterial cells. By investigating the single molecule dynamics of replicative helicase DnaC, DNA primase DnaG, and of lagging strand polymerase DnaE in the model bacterium Bacillus subtilis in response to transient replication blocks due to DNA damage, to inhibition of the replicative polymerase, or to downshift of serine availability, we show that proteins react differentially to the stress conditions. DnaG appears to be recruited to the forks by a diffusion and capture mechanism, becomes more statically associated after arrest of polymerase PolC, but binds much less often after fork blocks due to DNA damage or to nutritional downshift. These results indicate that binding of the alarmone ppGpp due to the stringent response prevents DnaG from binding to forks rather than blocking bound primase. Dissimilar behaviour of DnaG and of DnaE suggest that both proteins are recruited independently to the forks, rather than jointly. Turnover of all three proteins was increased during replication block after nutritional downshift, different from the situation due to DNA damage or polymerase inhibition, showing high plasticity of forks in response to different stress conditions. Forks persisted during all stress conditions, apparently ensuring rapid return to replication extension.

scholarly journals Symmetric activity of DNA polymerases at and recruitment of exonuclease ExoR and of PolA to the Bacillus subtilis replication forks

2019 ◽  
Vol 47 (16) ◽  
pp. 8521-8536 ◽  
Author(s):  
Rogelio Hernández-Tamayo ◽  
Luis M Oviedo-Bocanegra ◽  
Georg Fritz ◽  
Peter L Graumann
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Bacillus Subtilis ◽  
Single Molecule ◽  
Dna Polymerases ◽  
Replication Forks ◽  
Single Molecule Level ◽  
Time Dynamics ◽  
Bacterium Bacillus

AbstractDNA replication forks are intrinsically asymmetric and may arrest during the cell cycle upon encountering modifications in the DNA. We have studied real time dynamics of three DNA polymerases and an exonuclease at a single molecule level in the bacterium Bacillus subtilis. PolC and DnaE work in a symmetric manner and show similar dwell times. After addition of DNA damage, their static fractions and dwell times decreased, in agreement with increased re-establishment of replication forks. Only a minor fraction of replication forks showed a loss of active polymerases, indicating relatively robust activity during DNA repair. Conversely, PolA, homolog of polymerase I and exonuclease ExoR were rarely present at forks during unperturbed replication but were recruited to replications forks after induction of DNA damage. Protein dynamics of PolA or ExoR were altered in the absence of each other during exponential growth and during DNA repair, indicating overlapping functions. Purified ExoR displayed exonuclease activity and preferentially bound to DNA having 5′ overhangs in vitro. Our analyses support the idea that two replicative DNA polymerases work together at the lagging strand whilst only PolC acts at the leading strand, and that PolA and ExoR perform inducible functions at replication forks during DNA repair.

Multidimensional Characterization of Single‐Molecule Dynamics in a Plasmonic Nanocavity

2021 ◽  
Author(s):  
Lucas Domulevicz ◽  
Hyunhak Jeong ◽  
Nayan K. Paul ◽  
Juan Sebastian Gomez-Diaz ◽  
Joshua Hihath
Keyword(s):  
Single Molecule ◽  
Single Molecule Dynamics ◽  
Molecule Dynamics

scholarly journals Interactions and Localization of Escherichia coli Error-Prone DNA Polymerase IV after DNA Damage

2015 ◽  
Vol 197 (17) ◽  
pp. 2792-2809 ◽  
Author(s):  
Sarita Mallik ◽  
Ellen M. Popodi ◽  
Andrew J. Hanson ◽  
Patricia L. Foster
Keyword(s):  
Dna Damage ◽  
Dna Polymerase ◽  
Dna Helicase ◽  
Dna Lesions ◽  
Replication Forks ◽  
Content Type ◽  
Pol Iv ◽  
Dna Polymerase Iv ◽  
Stalled Replication Forks

ABSTRACTEscherichia coli's DNA polymerase IV (Pol IV/DinB), a member of the Y family of error-prone polymerases, is induced during the SOS response to DNA damage and is responsible for translesion bypass and adaptive (stress-induced) mutation. In this study, the localization of Pol IV after DNA damage was followed using fluorescent fusions. After exposure ofE. colito DNA-damaging agents, fluorescently tagged Pol IV localized to the nucleoid as foci. Stepwise photobleaching indicated ∼60% of the foci consisted of three Pol IV molecules, while ∼40% consisted of six Pol IV molecules. Fluorescently tagged Rep, a replication accessory DNA helicase, was recruited to the Pol IV foci after DNA damage, suggesting that thein vitrointeraction between Rep and Pol IV reported previously also occursin vivo. Fluorescently tagged RecA also formed foci after DNA damage, and Pol IV localized to them. To investigate if Pol IV localizes to double-strand breaks (DSBs), an I-SceI endonuclease-mediated DSB was introduced close to a fluorescently labeled LacO array on the chromosome. After DSB induction, Pol IV localized to the DSB site in ∼70% of SOS-induced cells. RecA also formed foci at the DSB sites, and Pol IV localized to the RecA foci. These results suggest that Pol IV interacts with RecAin vivoand is recruited to sites of DSBs to aid in the restoration of DNA replication.IMPORTANCEDNA polymerase IV (Pol IV/DinB) is an error-prone DNA polymerase capable of bypassing DNA lesions and aiding in the restart of stalled replication forks. In this work, we demonstratein vivolocalization of fluorescently tagged Pol IV to the nucleoid after DNA damage and to DNA double-strand breaks. We show colocalization of Pol IV with two proteins: Rep DNA helicase, which participates in replication, and RecA, which catalyzes recombinational repair of stalled replication forks. Time course experiments suggest that Pol IV recruits Rep and that RecA recruits Pol IV. These findings providein vivoevidence that Pol IV aids in maintaining genomic stability not only by bypassing DNA lesions but also by participating in the restoration of stalled replication forks.

Single-molecule dynamics of site-specific labeled transforming growth factor type II receptors on living cells

2014 ◽  
Vol 50 (94) ◽  
pp. 14724-14727 ◽  
Author(s):  
Ming Cheng ◽  
Wei Zhang ◽  
Jinghe Yuan ◽  
Wangxi Luo ◽  
Nan Li ◽  
...  
Keyword(s):  
Growth Factor ◽  
Single Molecule ◽  
Transforming Growth Factor ◽  
Living Cells ◽  
Unnatural Amino Acid ◽  
Type Ii ◽  
Site Specific ◽  
Single Molecule Dynamics ◽  
Factor Type ◽  
Molecule Dynamics

Single-molecule dynamics of the transforming growth factor type II receptor (TβRII) labeled by an unnatural amino acid.

Nanoconfinement greatly speeds up the nucleation and the annealing in single-DNA collapse

Soft Matter ◽  
2017 ◽  
Vol 13 (37) ◽  
pp. 6363-6371 ◽  
Author(s):  
Liang Dai ◽  
Jeremy J. Jones ◽  
Alexander R. Klotz ◽  
Stephen Levy ◽  
Patrick S. Doyle
Keyword(s):  
Single Molecule ◽  
Single Molecule Dynamics ◽  
New Phenomena ◽  
Molecule Dynamics

Manipulating and measuring single-molecule dynamics and reactions in nanofluidics is a rapidly growing field with broad applications in developing new biotechnologies, understanding nanoconfinement effects in vivo, and exploring new phenomena in confinement.

scholarly journals InferenceMAP: mapping of single-molecule dynamics with Bayesian inference

2015 ◽  
Vol 12 (7) ◽  
pp. 594-595 ◽  
Author(s):  
Mohamed El Beheiry ◽  
Maxime Dahan ◽  
Jean-Baptiste Masson
Keyword(s):  
Bayesian Inference ◽  
Single Molecule ◽  
Single Molecule Dynamics ◽  
Molecule Dynamics

Variable Temperature Single-Molecule Dynamics of MEH-PPV

ChemPhysChem ◽  
2005 ◽  
Vol 6 (11) ◽  
pp. 2404-2409 ◽  
Author(s):  
Young Jong Lee ◽  
Doo Young Kim ◽  
John K. Grey ◽  
Paul F. Barbara
Keyword(s):  
Single Molecule ◽  
Variable Temperature ◽  
Single Molecule Dynamics ◽  
Molecule Dynamics
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