Relative Contributions of Base Stacking and Electrostatic Repulsion on DNA Nicks and Gaps.

In duplex DNA, the continuous sugar phosphate backbones prevent the double helix from significant bending, but breaks in the duplex such as nicks, gaps, and flaps present points at which significant bending is possible. The conformational dynamics of these aberrant structures remains poorly understood. Two factors can maintain the duplexlike conformation of these aberrant structures, these being the hydrophobic and aromatic stacking interactions of the nucleobases, and the electrostatic repulsion of the negatively charged backbones. Using confocal single-molecule Förster resonance energy transfer on nicked and gapped DNA structures, we compare the relative contributions of these two factors by modulating the electrostatic repulsion through mono- and divalent cation concentrations. Base stacking interactions dominate the dynamics of nicked DNA, making it behave essentially like duplex DNA. Gapped structures have weaker base stacking and thus backbone electrostatic repulsion becomes important, and shielding from cations results in an average increase in bending around the gap. This bending of gapped structures could be interpreted by increased flexibility of unstacked structures, transient unstacking events, or a combination of the two. Burst variance analysis (BVA) and analysis by photon-by-photon hidden Markov modeling (H2MM), methods capable of detecting submillisecond dynamics of single molecules in solution, only revealed a single state, indicating that dynamics are occurring at time scales shorter than microseconds.

Single-molecule FRET unveils induced-fit mechanism for substrate selectivity in flap endonuclease 1.

Human flap endonuclease 1 (FEN1) and related structure-specific 5'nucleases precisely identify and incise aberrant DNA structures during replication, repair and recombination to avoid genomic instability. Yet, it is unclear how the 5'nuclease mechanisms of DNA distortion and protein ordering robustly mediate efficient and accurate substrate recognition and catalytic selectivity. Here, single-molecule sub-millisecond and millisecond analyses of FEN1 reveal a protein-DNA induced-fit mechanism that efficiently verifies substrate and suppresses off-target cleavage. FEN1 sculpts DNA with diffusion-limited kinetics to test DNA substrate. This DNA distortion mutually 'locks' protein and DNA conformation and enables substrate verification with extreme precision. Strikingly, FEN1 never misses cleavage of its cognate substrate while blocking probable formation of catalytically competent interactions with noncognate substrates and fostering their pre-incision dissociation. These findings establish FEN1 has practically perfect precision and that separate control of induced-fit substrate recognition sets up the catalytic selectivity of the nuclease active site for genome stability.

Noncontact, high-resolution ultrasonic imaging of wood samples using coded chirp waveforms.

A noncontact ultrasonic inspection technique has been developed to study the properties of wood samples in air. The system makes use of two broad bandwidth capacitive transducers, combined with signal processing techniques. A coded chirp signal was used in the current application to provide a waveform that could be postprocessed to provide sufficient sensitivity for transmission across samples of wood. It will be shown in this paper that the signal-to-noise ratio (SNR) can be greatly improved using two signal recovery techniques, namely pulse compression and swept frequency multiplication (SFM). A simulation of both techniques is presented and compared to experimental data. As will be seen from the experimental results, it is possible to perform noncontact ultrasonic experiments to extract a range of useful information such as ring density and the presence of microcracks.