Assembly mechanism and cryoEM structure of RecA recombination nucleofilaments from Streptococcus pneumoniae.

RecA-mediated homologous recombination (HR) is a key mechanism for genome maintenance and plasticity in bacteria. It proceeds through RecA assembly into a dynamic filament on ssDNA, the presynaptic filament, which mediates DNA homology search and ordered DNA strand exchange. Here, we combined structural, single molecule and biochemical approaches to characterize the ATP-dependent assembly mechanism of the presynaptic filament of RecA from Streptococcus pneumoniae (SpRecA), in comparison to the Escherichia coli RecA (EcRecA) paradigm. EcRecA polymerization on ssDNA is assisted by the Single-Stranded DNA Binding (SSB) protein, which unwinds ssDNA secondary structures that block EcRecA nucleofilament growth. We report by direct microscopic analysis of SpRecA filamentation on ssDNA that neither of the two paralogous pneumococcal SSBs could assist the extension of SpRecA nucleopolymers. Instead, we found that the conserved RadA helicase promotes SpRecA nucleofilamentation in an ATP-dependent manner. This allowed us to solve the atomic structure of such a long native SpRecA nucleopolymer by cryoEM stabilized with ATPγS. It was found to be equivalent to the crystal structure of the EcRecA filament with a marked difference in how RecA mediates nucleotide orientation in the stretched ssDNA. Then, our results show that SpRecA and EcRecA HR activities are different, in correlation with their distinct ATP-dependent ssDNA binding modes.

Digital One-Step Competitive Detection of a Small Molecule in Synthetic and Environmental Waters.

Optical methods for single-molecule analysis hold the promise of accurate, sensitive, and rapid detection of target molecules. Here, we demonstrate the efficiency of such an approach for the competitive detection of small molecules in water. Our biosensing method is based on a combination of a single-DNA biochip for the parallelization of tethered particle motion real-time measurements with antibodies and modified targets as molecular competitors. The antibodies are coupled to the particles tethered to the surface by a long DNA bearing in its middle the molecular competitor bound to the antibodies. Competitive target binding leads to a detectable conformational change of the DNA tethers from looped to unlooped in proportions related to the target concentration. We thus managed to detect fluorescein, chosen as a model of a target molecule, in freshwater of various qualities, from solutions prepared with ultrapure water to more complex matrices such as river water and wastewater treatment plant effluent samples. Similar dose-response curves were obtained under these various conditions in a wide range of concentrations from nanomolar to micromolar with a limit of detection around 2 nM.

Single-Molecule Sensing of DNA Intercalating Drugs in Water.

The occurrence of pharmaceutical residues in surface water is raising environmental concern. To accompany the evolution of measures for natural resources protection, sensing methods enabling sensitive and rapid water quality monitoring are needed. We recently managed the parallelization of the Tethered Particle Motion (TPM), a single molecule technique, sensitive to the conformational changes of DNA. Here, we investigate the capacity of high throughput TPM (htTPM) to detect drugs that intercalate into DNA. As a proof-of-concept we analyze the htTPM signal for two DNA intercalating dyes, namely, YOYO-1 and SYTOX orange. The efficient detection of intercalating drugs is then demonstrated with doxorubicin. We further evaluate the possibility to detect carbamazepine, an antiepileptic massively prescribed and persistent in water, which had been described to interact with DNA through intercalation. Our results corroborated by other techniques show that, in fact, carbamazepine is not a DNA intercalator. The comparison of the results obtained with different aqueous buffers and solutions allows us to identify optimal conditions for the monitoring of intercalation compounds by htTPM.