scholarly journals Single-molecule studies contrast ordered DNA replication with stochastic translesion synthesis

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Gengjing Zhao ◽  
Emma S Gleave ◽  
Meindert Hugo Lamers
Keyword(s):  
Dna Adducts ◽  
Single Molecule ◽  
Uv Light ◽  
Dna Polymerases ◽  
Stable Complex ◽  
High Fidelity ◽  
Single Molecule Spectroscopy ◽  
E Coli ◽  
Single Molecule Studies ◽  
Translesion Polymerases

High fidelity replicative DNA polymerases are unable to synthesize past DNA adducts that result from diverse chemicals, reactive oxygen species or UV light. To bypass these replication blocks, cells utilize specialized translesion DNA polymerases that are intrinsically error prone and associated with mutagenesis, drug resistance, and cancer. How untimely access of translesion polymerases to DNA is prevented is poorly understood. Here we use co-localization single-molecule spectroscopy (CoSMoS) to follow the exchange of the E. coli replicative DNA polymerase Pol IIIcore with the translesion polymerases Pol II and Pol IV. We find that in contrast to the toolbelt model, the replicative and translesion polymerases do not form a stable complex on one clamp but alternate their binding. Furthermore, while the loading of clamp and Pol IIIcore is highly organized, the exchange with the translesion polymerases is stochastic and is not determined by lesion-recognition but instead a concentration-dependent competition between the polymerases.

scholarly journals Single-Molecule Studies of the ssDNA Binding Activity of E. Coli MutL

2010 ◽  
Vol 98 (3) ◽  
pp. 64a-65a
Author(s):  
Jonghyun Park ◽  
Yong-Moon Jeon ◽  
Daekil In ◽  
Seong-Dal Heo ◽  
Changill Ban ◽  
...  
Keyword(s):  
Single Molecule ◽  
Binding Activity ◽  
Ssdna Binding ◽  
E Coli ◽  
Single Molecule Studies

scholarly journals A Primase-Induced Conformational Switch Controls the Stability of the Bacterial Replisome

2018 ◽  
Author(s):  
Enrico Monachino ◽  
Slobodan Jergic ◽  
Jacob S. Lewis ◽  
Zhi-Qiang Xu ◽  
Allen T.Y. Lo ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dna Polymerases ◽  
Fold Increase ◽  
Stable Complex ◽  
Conformational Switch ◽  
Bacterial Dna ◽  
Replicative Helicase ◽  
The Stability

SUMMARYRecent studies of bacterial DNA replication have led to a picture of the replisome as an entity that freely exchanges DNA polymerases and displays intermittent coupling between the helicase and polymerase(s). Challenging the textbook model of the polymerase holoenzyme acting as a stable complex coordinating the replisome, these observations suggest a role of the helicase as the central organizing hub. We show here that the molecular origin of this newly-found plasticity lies in the >400-fold increase in strength of the interaction between the polymerase holoenzyme and the replicative helicase upon association of the primase with the replisome. By combining in vitro ensemble-averaged and single-molecule assays, we demonstrate that this conformational switch operates during replication and promotes recruitment of multiple holoenzymes at the fork. Our observations provide a molecular mechanism for polymerase exchange and offer a revised model for the replication reaction that emphasizes its stochasticity.

scholarly journals Polymerization and editing modes of a high-fidelity DNA polymerase are linked by a well-defined path

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Thomas Dodd ◽  
Margherita Botto ◽  
Fabian Paul ◽  
Rafael Fernandez-Leiro ◽  
Meindert H. Lamers ◽  
...  
Keyword(s):  
Free Energy ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
High Fidelity ◽  
Exonuclease Activity ◽  
Strand Transfer ◽  
E Coli ◽  
Biochemical Analyses ◽  
Pol Iii ◽  
Fundamental Mechanism

Abstract Proofreading by replicative DNA polymerases is a fundamental mechanism ensuring DNA replication fidelity. In proofreading, mis-incorporated nucleotides are excised through the 3′-5′ exonuclease activity of the DNA polymerase holoenzyme. The exonuclease site is distal from the polymerization site, imposing stringent structural and kinetic requirements for efficient primer strand transfer. Yet, the molecular mechanism of this transfer is not known. Here we employ molecular simulations using recent cryo-EM structures and biochemical analyses to delineate an optimal free energy path connecting the polymerization and exonuclease states of E. coli replicative DNA polymerase Pol III. We identify structures for all intermediates, in which the transitioning primer strand is stabilized by conserved Pol III residues along the fingers, thumb and exonuclease domains. We demonstrate switching kinetics on a tens of milliseconds timescale and unveil a complete pol-to-exo switching mechanism, validated by targeted mutational experiments.

scholarly journals Facilitated Dissociation of Nucleoid Associated Proteins from DNA in the Bacterial Confinement

2021 ◽  
Author(s):  
Zafer Koşar ◽  
A. Göktuĝ Attar ◽  
Aykut Erbaş
Keyword(s):  
Dna Binding ◽  
Single Molecule ◽  
Experimental Studies ◽  
Dual Purpose ◽  
E Coli ◽  
Protein Dna Interactions ◽  
Factor For Inversion Stimulation ◽  
Associated Proteins ◽  
Order Of Magnitude ◽  
Single Molecule Studies

Transcription machinery ultimately depends on the temporal formation of protein-DNA complexes. Recent experimental studies demonstrate that residence time (i.e., inverse off-rate) of a transcription factor protein can be a contributor to the functional diversity of the protein. In the meantime, single-molecule experiments showed that the off-rates of a wide array of DNA-binding proteins accelerate as the bulk concentration of the protein increases via a concentration-dependent mechanism (i.e., facilitated dissociation, FD). In this study, inspired by the previous single-molecule studies on the factor for inversion stimulation (Fis) protein of E. coli, which is a dual-purpose protein with a diverse functionality, we model the unbinding of Fis from specific bindings sites along a high-molecular-weight circular DNA in a cylindrical structure mimicking the cellular confinement of chromosome. Our simulations show that FD of Fis can well occur in confinement at physiological concentrations. Particularly, when nutrient-rich conditions are emulated with Fis concentrations around micromolar levels, the off-rates increase one order of magnitude compared to the lower Fis levels. However, Fis significantly changes the chromosome structure at higher concentrations by forming dense protein clusters bridging specific sites and juxtaposing remote DNA segments. As a result, at the physiologically observed maximum levels of Fis, the off-rates significantly slow down. Overall, our results indicate that cellular-concentration levels of a structural DNA-binding protein is intermingled with the genome architecture and DNA residence times, thereby providing a basis for understanding the complex effects of dynamic protein-DNA interactions on gene regulation.

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