scholarly journals DNA mismatch and damage detection using a FRET-based assay for monitoring the loading of multiple MutS sliding clamps

2021 ◽  
Author(s):  
Vladislav Kunetsky ◽  
Olha Storozhuk ◽  
Gwendolyn Brouwer ◽  
Charlie Laffeber ◽  
Mark Simon Dillingham ◽  
...  
Keyword(s):  
Dna Damage ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Binding Activity ◽  
Bisulfite Conversion ◽  
Circular Dna ◽  
Alexa Fluor ◽  
Dna Mismatch ◽  
Sliding Clamps ◽  
Dna Mismatches

We developed a sensitive, homogeneous fluorescence assay for the detection of DNA mismatches and DNA damage based on the mismatch repair (MMR) protein MutS. The assay is based on Forster resonance energy transfer (FRET) between SYBR Green I (SG), non-covalently bound to DNA, and Alexa Fluor 647 (AF647) conjugated to MutS. In contrast to previous assays using only the mismatch binding activity of MutS, we exploited the ATP-dependent loading of multiple MutS sliding clamps provoked by mismatch/damage to the DNA, which increases the overall sensitivity of the assay. The assay was validated using a well-characterized 3 kb circular DNA containing a single G/T mismatch. We also demonstrate that treatment of long (multiple kb) DNA with various chemical or physical agents including non-denaturing bisulfite conversion of cytosine to uracil, cisplatin modification or ultraviolet light (UVC) results in changes in the DNA that can be detected by the FRET-based MutS biosensor.

scholarly journals Identification of the hydrophobic strand in the A–B loop of leptin as major binding site III: implications for large-scale preparation of potent recombinant human and ovine leptin antagonists

2005 ◽  
Vol 391 (2) ◽  
pp. 221-230 ◽  
Author(s):  
Leonora Niv-Spector ◽  
Dana Gonen-Berger ◽  
Isabelle Gourdou ◽  
Eva Biener ◽  
Eugene E. Gussakovsky ◽  
...  
Keyword(s):  
Amino Acids ◽  
Large Scale ◽  
Gel Filtration ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Binding Activity ◽  
Hydrophobic Cluster Analysis ◽  
Leptin Receptors ◽  
Subsequent Activation

Interaction of leptin with its receptors resembles that of interleukin-6 and granulocyte colony-stimulating factor, which interact with their receptors through binding sites I–III. Site III plays a pivotal role in receptors' dimerization or tetramerization and subsequent activation. Leptin's site III also mediates the formation of an active multimeric complex through its interaction with the IGD (immunoglobulin-like domain) of LEPRs (leptin receptors). Using a sensitive hydrophobic cluster analysis of leptin's and LEPR's sequences, we identified hydrophobic stretches in leptin's A–B loop (amino acids 39–42) and in the N-terminal end of LEPR's IGD (amino acids 325–328) that are predicted to participate in site III and to interact with each other in a β-sheet-like configuration. To verify this hypothesis, we prepared and purified to homogeneity (as verified by SDS/PAGE, gel filtration and reverse-phase chromatography) several alanine muteins of amino acids 39–42 in human and ovine leptins. CD analyses revealed that those mutations hardly affect the secondary structure. All muteins acted as true antagonists, i.e. they bound LEPR with an affinity similar to the wild-type hormone, had no agonistic activity and specifically inhibited leptin action in several leptin-responsive in vitro bioassays. Alanine mutagenesis of LEPR's IGD (amino acids 325–328) drastically reduced its biological but not binding activity, indicating the importance of this region for interaction with leptin's site III. FRET (fluorescence resonance energy transfer) microscopy experiments have documented that the transient FRET signalling occurring upon exposure to leptin results not from binding of the ligand, but from ligand-induced oligomerization of LEPRs mediated by leptin's site III.

scholarly journals Chromatin nanoscale compaction in live cells visualized by acceptor-donor ratio corrected FRET between DNA dyes

2019 ◽  
Author(s):  
Simone Pelicci ◽  
Alberto Diaspro ◽  
Luca Lanzanò
Keyword(s):  
Dna Damage ◽  
Energy Transfer ◽  
Fluorescence Lifetime ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Live Cells ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
Donor And Acceptor ◽  
Dna Dyes

AbstractChromatin nanoscale architecture in live cells can be studied by Forster Resonance Energy Transfer (FRET) between fluorescently labeled chromatin components, such as histones. A higher degree of nanoscale compaction is detected as a higher FRET level, since this corresponds to a higher degree of proximity between donor and acceptor molecules. However, in such a system the stoichiometry of the donors and acceptors engaged in the FRET process is not well defined and, in principle, FRET variations could be caused by variations in the acceptor-donor ratio rather than distance. Here we show that a FRET value independent of the acceptor-donor ratio can be obtained by Fluorescence Lifetime Imaging (FLIM) detection of FRET combined with a normalization of the FRET level to a pixel-wise estimation of the acceptor-donor ratio. We use this method to study FRET between two DNA binding dyes staining the nuclei of live cells. We show that acceptor-donor ratio corrected FRET imaging reveals variations of nanoscale compaction in different chromatin environments. As an application, we monitor the rearrangement of chromatin in response to laser-induced micro-irradiation and reveal that DNA is rapidly decompacted, at the nanoscale, in response to DNA damage induction.

DNA Mismatch Detection by Resonance Energy Transfer between Ruthenium(II) and Osmium(II) Tris(2,2‘-bipyridyl) Chromophores

2005 ◽  
Vol 44 (12) ◽  
pp. 4112-4114 ◽  
Author(s):  
Elena V. Bichenkova ◽  
Xuan Yu ◽  
Pranab Bhadra ◽  
Helena Heissigerova ◽  
Simon J. A. Pope ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Dna Mismatch ◽  
Mismatch Detection

Electrochemiluminescence resonance energy transfer between graphene quantum dots and gold nanoparticles for DNA damage detection

The Analyst ◽  
2014 ◽  
Vol 139 (10) ◽  
pp. 2404-2410 ◽  
Author(s):  
Qian Lu ◽  
Wei Wei ◽  
Zhenxian Zhou ◽  
Zhixin Zhou ◽  
Yuanjian Zhang ◽  
...  
Keyword(s):  
Dna Damage ◽  
Quantum Dots ◽  
Gold Nanoparticles ◽  
Energy Transfer ◽  
Damage Detection ◽  
Au Nanoparticles ◽  
Graphene Quantum Dots ◽  
Resonance Energy Transfer ◽  
Resonance Energy

Electrochemiluminescence resonance energy transfer between graphene quantum dots (GQDs) and Au nanoparticles results in the electrochemiluminescence signal of the GQDs being quenched or recovering.

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.

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