scholarly journals Probing the molecular mechanism of action of Deinococcus radiodurans RecD2 at single-molecule resolution

2021 ◽  
Author(s):  
Deb Purkait ◽  
Farhana Islam ◽  
Padmaja P. Mishra
Keyword(s):  
Single Molecule ◽  
Deinococcus Radiodurans ◽  
Excision Repair ◽  
Structural Characteristics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Dna Unwinding ◽  
X Ray Crystallography ◽  
Domains Of Life ◽  
Molecular Mechanism Of Action

Helicases are ATP-driven molecular machines that directionally remodel nucleic acid polymers in all three domains of life. Helicases are responsible for resolving double-stranded DNA (dsDNA) into separate single-strands and this activity is essential for DNA replication, nucleotide excision repair, and homologous recombination. RecD2 from Deinococcus radiodurans (DrRecD2) has important contributions towards its unusually high tolerance to gamma radiation and hydrogen peroxide. Although previous X-ray Crystallography studies have revealed the structural characteristics of the protein, the direct experimental evidence regarding the dynamics of the DNA unwinding process by DrRecD2 in the context of other accessory proteins is yet to be found. In this study, we have probed the exact binding event and processivity of DrRecD2 at single-molecule resolution using Protein-induced fluorescence enhancement (smPIFE) and Forster resonance energy transfer (smFRET). We have found that the protein prefers to bind at the 5 prime terminal end of the single-stranded DNA (ssDNA) by Drift and has helicase activity even in absence of ATP. However, a faster and iterative mode of DNA unwinding was evident in presence of ATP. The rate of translocation of the protein was found to be slower on dsDNA compared to ssDNA. We also showed that DrRecD2 is recruited at the binding site by the single-strand binding protein (SSB) and during the unwinding, it can displace RecA from ssDNA.

scholarly journals The mechanism of gap creation by a multifunctional nuclease during base excision repair

2021 ◽  
Vol 7 (29) ◽  
pp. eabg0076
Author(s):  
Jungmin Yoo ◽  
Donghun Lee ◽  
Hyeryeon Im ◽  
Sangmi Ji ◽  
Sanghoon Oh ◽  
...  
Keyword(s):  
Base Excision Repair ◽  
Single Molecule ◽  
Excision Repair ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Ap Endonuclease ◽  
Base Excision ◽  
Polymerase I ◽  
Gap Creation ◽  
Ap Site

During base excision repair, a transient single-stranded DNA (ssDNA) gap is produced at the apurinic/apyrimidinic (AP) site. Exonuclease III, capable of performing both AP endonuclease and exonuclease activity, are responsible for gap creation in bacteria. We used single-molecule fluorescence resonance energy transfer to examine the mechanism of gap creation. We found an AP site anchor-based mechanism by which the intrinsically distributive enzyme binds strongly to the AP site and becomes a processive enzyme, rapidly creating a gap and an associated transient ssDNA loop. The gap size is determined by the rigidity of the ssDNA loop and the duplex stability of the DNA and is limited to a few nucleotides to maintain genomic stability. When the 3′ end is released from the AP endonuclease, polymerase I quickly initiates DNA synthesis and fills the gap. Our work provides previously unidentified insights into how a signal of DNA damage changes the enzymatic functions.

scholarly journals RNA polymerase clamp conformational dynamics: long-lived states and modulation by crowding, cations, and nonspecific DNA binding

2021 ◽  
Author(s):  
Abhishek Mazumder ◽  
Anna Wang ◽  
Heesoo Uhm ◽  
Richard H Ebright ◽  
Achillefs N Kapanidis
Keyword(s):  
Rna Polymerase ◽  
Small Molecules ◽  
Single Molecule ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Monovalent Cation ◽  
Structural Element ◽  
Conformational States ◽  
Domains Of Life

Abstract The RNA polymerase (RNAP) clamp, a mobile structural element conserved in RNAP from all domains of life, has been proposed to play critical roles at different stages of transcription. In previous work, we demonstrated using single-molecule Förster resonance energy transfer (smFRET) that RNAP clamp interconvert between three short-lived conformational states (lifetimes ∼ 0.3–0.6 s), that the clamp can be locked into any one of these states by small molecules, and that the clamp stays closed during initial transcription and elongation. Here, we extend these studies to obtain a comprehensive understanding of clamp dynamics under conditions RNAP may encounter in living cells. We find that the RNAP clamp can populate long-lived conformational states (lifetimes > 1.0 s) and can switch between these long-lived states and the previously observed short-lived states. In addition, we find that clamp motions are increased in the presence of molecular crowding, are unchanged in the presence of elevated monovalent-cation concentrations, and are reduced in the presence of elevated divalent-cation concentrations. Finally, we find that RNAP bound to non-specific DNA predominantly exhibits a closed clamp conformation. Our results raise the possibility of additional regulatory checkpoints that could affect clamp dynamics and consequently could affect transcription and transcriptional regulation.

scholarly journals DNA Breaks and Gaps Target Retroviral Integration

2021 ◽  
Author(s):  
Gayan Senavirathne ◽  
Anne Gardner ◽  
James London ◽  
Ryan K. Messer ◽  
Yow-Yong Tan ◽  
...  
Keyword(s):  
Single Molecule ◽  
Excision Repair ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Dna Breaks ◽  
Retroviral Integration ◽  
Dna Lesion ◽  
Nucleotide Insertion ◽  
Base Excision

Integration into a host genome is essential for retrovirus infection and is catalyzed by a nucleoprotein complex (Intasome) containing the virus-encoded integrase (IN) and the reverse transcribed (RT) virus copy DNA (cDNA). Previous studies suggested that integration was limited by intasome-host DNA recognition progressions. Using single molecule Forster resonance energy transfer (smFRET) we show that PFV intasomes pause at nicked and gapped DNA, which targeted site-directed integration without inducing significant intasome conformational alterations. Base excision repair (BER) components that affect retroviral integration in vivo produce similar nick/gap intermediates during DNA lesion processing. Intasome pause dynamics was modified by the 5′-nick-gap chemistry, while an 8-oxo-guanine lesion, a mismatch, or a nucleotide insertion that induce backbone flexibility and/or static bends had no effect. These results suggest that dynamic often non-productive intasome-DNA interactions may be modulated to target retroviral integration.

scholarly journals RNA polymerase clamp conformational dynamics: long-lived states and modulation by crowding, cations, and nonspecific DNA binding

2020 ◽  
Author(s):  
Abhishek Mazumder ◽  
Anna Wang ◽  
Heesoo Uhm ◽  
Richard H. Ebright ◽  
Achillefs N. Kapanidis
Keyword(s):  
Rna Polymerase ◽  
Small Molecules ◽  
Single Molecule ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Monovalent Cation ◽  
Structural Element ◽  
Conformational States ◽  
Domains Of Life

AbstractThe RNA polymerase (RNAP) clamp, a mobile structural element conserved in RNAP from all domains of life, has been proposed to play critical roles at different stages of transcription. In previous work, we demonstrated using single-molecule Förster resonance energy transfer (smFRET) that RNAP clamp interconvert between three short-lived conformational states (lifetimes ∼ 0.3-0.6 s), that the clamp can be locked into any one of these states by small molecules, and that the clamp stays closed during initial transcription and elongation. Here, we extend these studies to obtain a comprehensive understanding of clamp dynamics under conditions RNAP may encounter in living cells. We find that the RNAP clamp can populate long-lived conformational states (lifetimes >1.0 s) and can switch between these long-lived states and the previously observed short-lived states. In addition, we find that clamp motions are increased in the presence of molecular crowding, are unchanged in the presence of elevated monovalent-cation concentrations, and are reduced in the presence of elevated divalent-cation concentrations. Finally, we find that RNAP bound to non-specific DNA predominantly exhibits a closed clamp conformation. Our results raise the possibility of additional regulatory checkpoints that could affect clamp dynamics and consequently could affect transcription and transcriptional regulation.

Structural dynamics of P-type ATPase ion pumps

2019 ◽  
Vol 47 (5) ◽  
pp. 1247-1257 ◽  
Author(s):  
Mateusz Dyla ◽  
Sara Basse Hansen ◽  
Poul Nissen ◽  
Magnus Kjaergaard
Keyword(s):  
Structural Dynamics ◽  
Single Molecule ◽  
New Technologies ◽  
Atp Hydrolysis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Resolved ◽  
Dynamics Simulations ◽  
Types Of Information ◽  
P Type

Abstract P-type ATPases transport ions across biological membranes against concentration gradients and are essential for all cells. They use the energy from ATP hydrolysis to propel large intramolecular movements, which drive vectorial transport of ions. Tight coordination of the motions of the pump is required to couple the two spatially distant processes of ion binding and ATP hydrolysis. Here, we review our current understanding of the structural dynamics of P-type ATPases, focusing primarily on Ca2+ pumps. We integrate different types of information that report on structural dynamics, primarily time-resolved fluorescence experiments including single-molecule Förster resonance energy transfer and molecular dynamics simulations, and interpret them in the framework provided by the numerous crystal structures of sarco/endoplasmic reticulum Ca2+-ATPase. We discuss the challenges in characterizing the dynamics of membrane pumps, and the likely impact of new technologies on the field.

Monitoring the Structural Dynamics of U2 snRNA Via Single-Molecule Förster Resonance Energy Transfer

2018 ◽  
Author(s):  
Alexander Carl DeHaven
Keyword(s):  
Energy Transfer ◽  
Structural Dynamics ◽  
Single Molecule ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Förster Resonance Energy Transfer ◽  
U2 Snrna ◽  
Folding Dynamics ◽  
The Impact

This thesis contains four topic areas: a review of single-molecule microscropy methods and splicing, conformational dynamics of stem II of the U2 snRNA, the impact of post-transcriptional modifications on U2 snRNA folding dynamics, and preliminary findings on Mango aptamer folding dynamics.

Towards Single-Molecule Diagnostics Using Microfluidic Manipulation and Quantum Dot Nanosensors

2007 ◽  
Author(s):  
Hsin-Chih Yeh ◽  
Christopher M. Puleo ◽  
Yi-Ping Ho ◽  
Tza-Huei Wang
Keyword(s):  
Energy Transfer ◽  
Quantum Dot ◽  
Single Molecule ◽  
Dna Sequences ◽  
Mutational Analysis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Single Molecule Detection ◽  
Specific Analysis ◽  
Low Volume

In this report, we review several single-molecule detection (SMD) methods and newly developed nanocrystal-mediated single-fluorophore strategies for ultrasensitive and specific analysis of genomic sequences. These include techniques, such as quantum dot (QD)-mediated fluorescence resonance energy transfer (FRET) technology and dual-color fluorescence coincidence and colocalization analysis, which allow separation-free detection of low-abundance DNA sequences and mutational analysis of oncogenes. Microfluidic approaches developed for use with single-molecule detection to achieve rapid, low-volume, and quantitative analysis of nucleic acids, such as electrokinetic manipulation of single molecules and confinement of sub-nanoliter samples using microfluidic networks integrated with valves, are also discussed.

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