How to Quantify DNA Compaction by TFAM with Acoustic Force Spectroscopy and Total Internal Reflection Fluorescence Microscopy

Martucci, Martial; Debar, Louis; Wildenberg, Siet van den; Farge, Géraldine

doi:10.1007/978-1-0716-2922-2_10

Cited by 3 publications

(4 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Knowing that tetramer destabilization and binding to short ssDNA templates are not the molecular mechanisms explaining the R107Q phenotype, we then investigated the interaction of mtSSB with longer, more physiological, ssDNA substrates. For this, we used an inverted fluorescence microscope in combination with a camera which allows imaging of a large field of view at the cost of single-molecule resolution (for details see ( 39 )). We first visualized the effect of mtSSB on DNA compaction.…”

Section: Resultsmentioning

confidence: 99%

“…The TIRF experimental setup was described in detail in ( 39 ). For fluorescent imaging of mtSSB Alexa555 and mtSSB Alexa647 , a 532-nm and 640-nm excitation lasers were used and imaged either on a Nikon (DS-Qi2) wide field camera (compaction assay) or on an EMCCD (iXon 888, ANDOR) camera (dissociation and competition assays).…”

Section: Methodsmentioning

confidence: 99%

“…For AFS experiments, DNA tethers were made by attaching DNA molecules labelled on one end with digoxigenin and on the other end with biotins between a glass surface and 4.89 μm streptavidin polystyrene beads (Spherotech). A detailed description of the AFS instrumentation (LUMICKS B.V., Amsterdam, the Netherlands) can be found in ( 39 ). Flow-cell functionalization was performed with anti-digoxigenin (50 μg/ml, Roche) for 20 min and passivation with PBS containing 2% BSA for at least 30 min.…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

The mutation R107Q alters mtSSB ssDNA compaction ability and binding dynamics

Martucci,

Moretton,

Tarrés-Solé

et al. 2024

Nucleic Acids Research

Self Cite

View full text Add to dashboard Cite

Mitochondrial single-stranded DNA-binding protein (mtSSB) is essential for mitochondrial DNA (mtDNA) replication. Recently, several mtSSB variants have been associated with autosomal dominant mitochondrial optic atrophy and retinal dystrophy. Here, we have studied at the molecular level the functional consequences of one of the most severe mtSSB variants, R107Q. We first studied the oligomeric state of this variant and observed that the mtSSBR107Q mutant forms stable tetramers in vitro. On the other hand, we showed, using complementary single-molecule approaches, that mtSSBR107Q displays a lower intramolecular ssDNA compaction ability and a higher ssDNA dissociation rate than the WT protein. Real-time competition experiments for ssDNA-binding showed a marked advantage of mtSSBWT over mtSSBR107Q. Combined, these results show that the R107Q mutation significantly impaired the ssDNA-binding and compacting ability of mtSSB, likely by weakening mtSSB ssDNA wrapping efficiency. These features are in line with our molecular modeling of ssDNA on mtSSB showing that the R107Q mutation may destabilize local interactions and results in an electronegative spot that interrupts an ssDNA-interacting-electropositive patch, thus reducing the potential mtSSB-ssDNA interaction sites.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

The mutation R107Q alters mtSSB ssDNA compaction ability and binding dynamics

Martucci,

Moretton,

Tarrés-Solé

et al. 2024

Nucleic Acids Research

Self Cite

View full text Add to dashboard Cite

show abstract

“…Biological processes such as receptor mediated signaling, cell migration, mechanotransduction, and muscle function often involve generation and detection of pico-to nano-Newton level forces (1)(2)(3)(4)(5)(6)(7). Singlemolecule force spectroscopy techniques such as atomic force microscopy (AFM), optical tweezers, magnetic tweezers, and acoustic force spectroscopy are well suited to study force-induced structural changes in biomolecular structures in vitro (8)(9)(10)(11)(12). However, these techniques use perturbing external forces as well as micron-scale probes such as beads or functionalized cantilevers that can limit their ability to characterize biological processes in situ, for example in live cell assays.…”

Section: Introductionmentioning

confidence: 99%

In-SilicoAnalyses of Molecular Force Sensors for Mechanical Characterization of Biological Systems

Lopez,

Castro,

Sotomayor

2024

Preprint

View full text Add to dashboard Cite

Mechanical forces play key roles in biological processes such as cell migration and sensory perception. In recent years molecular force sensors have been developed as tools for in situ force measurements. Here we use all-atom steered molecular dynamics simulations to predict and study the relationship between design parameters and mechanical properties for three types of molecular force sensors commonly used in cellular biological research: two peptide- and one DNA-based. The peptide-based sensors consist of a pair of fluorescent proteins, which can undergo Forster resonance energy transfer (FRET), linked by spider silk (GPGGA)n or synthetic (GGSGGS)n disordered regions. The DNA-based sensor consists of two fluorophore-labeled strands of DNA that can be unzipped or sheared upon force application with a FRET signal as readout of dissociation. We simulated nine sensors, three of each kind. After equilibration, flexible peptide linkers of three different lengths were stretched by applying forces to their N- and C-terminal Calpha atoms in opposite directions. Similarly, we equilibrated a DNA-based sensor and pulled on the phosphate atom of the terminal guanine of one strand and a selected phosphate atom on the other strand in the opposite direction. These simulations were performed at constant velocity (0.01 nm/ns - 10 nm/ns) and constant force (10 pN - 500 pN) for all versions of the sensors. Our results show how the force response of these sensors depends on their length, sequence, configuration and loading rate. Mechanistic insights gained from simulations analyses indicate that interpretation of experimental results should consider the influence of transient formation of secondary structure in peptide-based sensors and of overstretching in DNA-based sensors. These predictions can guide optimal fluorophore choice and facilitate the rational design of new sensors for use in protein, DNA, hybrid systems, and molecular devices.

show abstract