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 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 Rational design of protein-specific folding modifiers

2020 ◽  
Author(s):  
Anirban Das ◽  
Anju Yadav ◽  
Mona Gupta ◽  
R Purushotham ◽  
Vishram L. Terse ◽  
...  
Keyword(s):  
Single Molecule ◽  
Rational Design ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Force Measurements ◽  
Folding Nucleus ◽  
Dynamics Simulations ◽  
General Method

AbstractProtein folding can go wrong in vivo and in vitro, with significant consequences for the living cell and the pharmaceutical industry, respectively. Here we propose a general design principle for constructing small peptide-based protein-specific folding modifiers. We construct a ‘xenonucleus’, which is a pre-folded peptide that resembles the folding nucleus of a protein, and demonstrate its activity on the folding of ubiquitin. Using stopped-flow kinetics, NMR spectroscopy, Förster Resonance Energy transfer, single-molecule force measurements, and molecular dynamics simulations, we show that the ubiquitin xenonucleus can act as an effective decoy for the native folding nucleus. It can make the refolding faster by 33 ± 5% at 3 M GdnHCl. In principle, our approach provides a general method for constructing specific, genetically encodable, folding modifiers for any protein which has a well-defined contiguous folding nucleus.

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 Lateral gate dynamics of the bacterial translocon during cotranslational membrane protein insertion

2021 ◽  
Vol 118 (26) ◽  
pp. e2100474118
Author(s):  
Evan Mercier ◽  
Xiaolin Wang ◽  
Manisankar Maiti ◽  
Wolfgang Wintermeyer ◽  
Marina V. Rodnina
Keyword(s):  
Membrane Proteins ◽  
Single Molecule ◽  
Biological Significance ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Phospholipid Bilayer ◽  
Membrane Phospholipids ◽  
Model Free ◽  
Lateral Gate

During synthesis of membrane proteins, transmembrane segments (TMs) of nascent proteins emerging from the ribosome are inserted into the central pore of the translocon (SecYEG in bacteria) and access the phospholipid bilayer through the open lateral gate formed of two helices of SecY. Here we use single-molecule fluorescence resonance energy transfer to monitor lateral-gate fluctuations in SecYEG embedded in nanodiscs containing native membrane phospholipids. We find the lateral gate to be highly dynamic, sampling the whole range of conformations between open and closed even in the absence of ligands, and we suggest a statistical model-free approach to evaluate the ensemble dynamics. Lateral gate fluctuations take place on both short (submillisecond) and long (subsecond) timescales. Ribosome binding and TM insertion do not halt fluctuations but tend to increase sampling of the open state. When YidC, a constituent of the holotranslocon, is bound to SecYEG, TM insertion facilitates substantial opening of the gate, which may aid in the folding of YidC-dependent polytopic membrane proteins. Mutations in lateral gate residues showing in vivo phenotypes change the range of favored states, underscoring the biological significance of lateral gate fluctuations. The results suggest how rapid fluctuations of the lateral gate contribute to the biogenesis of inner-membrane proteins.

scholarly journals Nanomechanics and co-transcriptional folding of Spinach and Mango

2019 ◽  
Author(s):  
Jaba Mitra ◽  
Taekjip Ha
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Mechanical Stability ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Rna Aptamers ◽  
Cellular Processes ◽  
Force Relaxation ◽  
And Function

AbstractRecent advances in fluorogen-binding RNA aptamers known as “light-up” aptamers provide an avenue for protein-free detection of RNA in cells. Crystallographic studies have revealed a G-Quadruplex (GQ) structure at the core of light-up aptamers such as Spinach, Mango and Corn. Detailed biophysical characterization of folding of such aptamers is still lacking despite the potential implications on their in vivo folding and function. We used single-molecule fluorescence-force spectroscopy that combines fluorescence resonance energy transfer with optical tweezers to examine mechanical responses of Spinach2, iMangoIII and MangoIV. Spinach2 unfolded in four discrete steps as force is increased to 7 pN and refolded in reciprocal steps upon force relaxation. Binding of DFHBI-1T fluorogen preserved the step-wise unfolding behavior although at slightly higher forces. In contrast, GQ core unfolding in iMangoIII and MangoIV occurred in one discrete step at forces > 10 pN and refolding occurred at lower forces showing hysteresis. Binding of the cognate fluorogen, TO1, did not significantly alter the mechanical stability of Mangos. In addition to K+, which is needed to stabilize the GQ cores, Mg2+ was needed to obtain full mechanical stability of the aptamers. Co-transcriptional folding analysis using superhelicases showed that co-transcriptional folding reduces misfolding and allows a folding pathway different from refolding. As the fundamental cellular processes like replication, transcription etc. exert pico-Newton levels of force, these aptamers may unfold in vivo and subsequently misfold.

scholarly journals Optimal molecular crowding accelerates group II intron folding and maximizes catalysis

2018 ◽  
Vol 115 (47) ◽  
pp. 11917-11922 ◽  
Author(s):  
Bishnu P. Paudel ◽  
Erica Fiorini ◽  
Richard Börner ◽  
Roland K. O. Sigel ◽  
David S. Rueda
Keyword(s):  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Group Ii Intron ◽  
Maximum Activity ◽  
Molecular Crowding ◽  
Cellular Environment ◽  
Group Ii

Unlike in vivo conditions, group II intron ribozymes are known to require high magnesium(II) concentrations ([Mg2+]) and high temperatures (42 °C) for folding and catalysis in vitro. A possible explanation for this difference is the highly crowded cellular environment, which can be mimicked in vitro by macromolecular crowding agents. Here, we combined bulk activity assays and single-molecule Förster Resonance Energy Transfer (smFRET) to study the influence of polyethylene glycol (PEG) on catalysis and folding of the ribozyme. Our activity studies reveal that PEG reduces the [Mg2+] required, and we found an “optimum” [PEG] that yields maximum activity. smFRET experiments show that the most compact state population, the putative active state, increases with increasing [PEG]. Dynamic transitions between folded states also increase. Therefore, this study shows that optimal molecular crowding concentrations help the ribozyme not only to reach the native fold but also to increase its in vitro activity to approach that in physiological conditions.

scholarly journals XRCC1 Is Specifically Associated with Poly(ADP-Ribose) Polymerase and Negatively Regulates Its Activity following DNA Damage

1998 ◽  
Vol 18 (6) ◽  
pp. 3563-3571 ◽  
Author(s):  
Murielle Masson ◽  
Claude Niedergang ◽  
Valérie Schreiber ◽  
Sylviane Muller ◽  
Josiane Menissier-de Murcia ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Mammalian Cells ◽  
Excision Repair ◽  
Cell Cycle Checkpoint ◽  
Dna Breaks ◽  
Protective Function ◽  
C Terminus ◽  
Base Excision

ABSTRACT Poly(ADP-ribose) polymerase (PARP; EC 2.4.2.30 ) is a zinc-finger DNA-binding protein that detects and signals DNA strand breaks generated directly or indirectly by genotoxic agents. In response to these breaks, the immediate poly(ADP-ribosyl)ation of nuclear proteins involved in chromatin architecture and DNA metabolism converts DNA damage into intracellular signals that can activate DNA repair programs or cell death options. To have greater insight into the physiological function of this enzyme, we have used the two-hybrid system to find genes encoding proteins putatively interacting with PARP. We have identified a physical association between PARP and the base excision repair (BER) protein XRCC1 (X-ray repair cross-complementing 1) in theSaccharomyces cerevisiae system, which was further confirmed to exist in mammalian cells. XRCC1 interacts with PARP by its central region (amino acids 301 to 402), which contains a BRCT (BRCA1 C terminus) module, a widespread motif in DNA repair and DNA damage-responsive cell cycle checkpoint proteins. Overexpression of XRCC1 in Cos-7 or HeLa cells dramatically decreases PARP activity in vivo, reinforcing the potential protective function of PARP at DNA breaks. Given that XRCC1 is also associated with DNA ligase III via a second BRCT module and with DNA polymerase β, our results provide strong evidence that PARP is a member of a BER multiprotein complex involved in the detection of DNA interruptions and possibly in the recruitment of XRCC1 and its partners for efficient processing of these breaks in a coordinated manner. The modular organizations of these interactors, associated with small conserved domains, may contribute to increasing the efficiency of the overall pathway.

scholarly journals Endonuclease IV Is the Main Base Excision Repair Enzyme Involved in DNA Damage Induced by UVA Radiation and Stannous Chloride

2010 ◽  
Vol 2010 ◽  
pp. 1-9 ◽  
Author(s):  
Ellen S. Motta ◽  
Paulo Thiago Souza-Santos ◽  
Tuany R. Cassiano ◽  
Flávio J. S. Dantas ◽  
Adriano Caldeira-de-Araujo ◽  
...  
Keyword(s):  
Stannous Chloride ◽  
Excision Repair ◽  
Dna Lesions ◽  
Dna Breaks ◽  
Strand Breaks ◽  
E Coli ◽  
Dna Lesion ◽  
Base Excision ◽  
Dna Strand ◽  
Uva Radiation

Stannous chloride (SnCl2) and UVA induce DNA lesions through ROS. The aim of this work was to study the toxicity induced by UVA preillumination, followed bySnCl2treatment.E. coliBER mutants were used to identify genes which could play a role in DNA lesion repair generated by these agents. The survival assays showed (i) Thenfomutant was the most sensitive toSnCl2; (ii) lethal synergistic effect was observed after UVA pre-illumination, plusSnCl2incubation, thenfomutant being the most sensitive; (iii) wild type andnfomutants, transformed with pBW21 plasmid (nfo+) had their survival increased following treatments. The alkaline agarose gel electrophoresis assays pointed that (i) UVA induced DNA breaks andfpgmutant was the most sensitive; (ii)SnCl2-induced DNA strand breaks were higher than those from UVA andnfomutant had the slowest repair kinetics; (iii)UVA+SnCl2promoted an increase in DNA breaks thanSnCl2and, again,nfomutant displayed the slowest repair kinetics. In summary, Nfo protectsE. colicells against damage induced bySnCl2andUVA+SnCl2.

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.

Programmable in vitro Co-Encapsidation of Guest Proteins Inside Protein Nanocages

2018 ◽  
Author(s):  
Noor H. Dashti ◽  
Rufika S. Abidin ◽  
Frank Sainsbury
Keyword(s):  
Self Assembly ◽  
Mammalian Cells ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Super Resolution ◽  
Cell Engineering ◽  
Particle Analysis ◽  
Protein Cages

Bioinspired self-sorting and self-assembling systems using engineered versions of natural protein cages have been developed for biocatalysis and therapeutic delivery. The packaging and intracellular delivery of guest proteins is of particular interest for both <i>in vitro</i> and <i>in vivo</i> cell engineering. However, there is a lack of platforms in bionanotechnology that combine programmable guest protein encapsidation with efficient intracellular uptake. We report a minimal peptide anchor for <i>in vivo</i> self-sorting of cargo-linked capsomeres of the Murine polyomavirus (MPyV) major coat protein that enables controlled encapsidation of guest proteins by <i>in vitro</i> self-assembly. Using Förster resonance energy transfer (FRET) we demonstrate the flexibility in this system to support co-encapsidation of multiple proteins. Complementing these ensemble measurements with single particle analysis by super-resolution microscopy shows that the stochastic nature of co-encapsidation is an overriding principle. This has implications for the design and deployment of both native and engineered self-sorting encapsulation systems and for the assembly of infectious virions. Taking advantage of the encoded affinity for sialic acids ubiquitously displayed on the surface of mammalian cells, we demonstrate the ability of self-assembled MPyV virus-like particles to mediate efficient delivery of guest proteins to the cytosol of primary human cells. This platform for programmable co-encapsidation and efficient cytosolic delivery of complementary biomolecules therefore has enormous potential in cell engineering.

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