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.DOI: http://dx.doi.org/10.7554/eLife.21884.001

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Single molecule Förster resonance energy transfer (smFRET) is a unique biophysical approach for studying conformational dynamics in biomacromolecules. Photon-by-photon hidden Markov modeling (H2MM) is an analysis tool that can quantify FRET dynamics of single biomolecules, even if they occur on the sub-millisecond timescale. However, dye photophysical transitions intertwined with FRET dynamics may cause artifacts. Here, we introduce multi-parameter H2MM (mpH2MM), which assists in identifying FRET dynamics based on simultaneous observation of multiple experimentally-derived parameters. We show the importance of using mpH2MM to decouple FRET dynamics caused by conformational changes from photophysical transitions in confocal-based smFRET measurements of a DNA hairpin, the maltose binding protein, MalE, and the type-III secretion system effector, YopO, from Yersinia species, all exhibiting conformational dynamics ranging from the sub-second to microsecond timescales. Overall, we show that using mpH2MM facilitates the identification and quantification of biomolecular sub-populations and their origin.

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It is generally accepted that the tone of the peripheral arterioles plays an important part in regulating the systemic blood pressure in man. Whether the small vessels of the lungs exercise similar control over the pressure in the pulmonary artery is not so certain. This uncertainty has stemmed largely from the fact that the human pulmonary vessels have exhibited an erratic response to many vasoactive drugs (1-15). As a consequence, most physiologists have concluded either that the pulmonary vessels are incapable of intrinsic changes in tone, or that the effect of such changes, if they occur, is less important in determining the pulmonary arterial pressure than is the effect of mechanical factors alone.There is, however, considerable evidence to suggest that acute hypoxia increases the pulmonary vascular tone (16)(17)(18)(19)(20). This stimulus raises the pulmonary arterial pressure by a greater amount than might be expected to result from the increase in blood flow which occurs. Moreover, the pulmonary wedge and systemic pressures are not altered (19), and there is no consistent variation in the central blood volume (19,21). From these observations it may be inferred that hypoxia constricts the vessels of the lungs.Active dilatation of these vessels has not been so adequately demonstrated. Most drugs which lower the pulmonary arterial pressure have a concomitant action on the systemic pressure, and it 1 This investigation was supported by a research grant [H-2001 (C) ] from the National Heart Institute of the is difficult to determine whether the changes in the pulmonary circulation represent a primary or a secondary effect. However, one of us (P.H.) observed that a single dose of acetylcholine can lower the pulmonary arterial pressure without affecting the pressure in the systemic vessels (22,23). The response was transient, and was found only in patients with a moderate degree of pulmonary hypertension.Since it seemed possible that this fall in pressure occurred as a result of active vasodilatation, the present project was undertaken with two objectives in view: 1) to investigate the effect of a continuous infusion of acetylcholine into the pulmonary artery of normal subjects, and 2) to inquire whether the action of the drug was enhanced when the pulmonary arterial pressure in these same subjects had been raised by making them hypoxic. METHODSEach subject was studied in the unanesthetized basal state. Respiratory studies were carried out on a previous day to acquaint the subject with the laboratory personnel and to accustom him into the effects of breathing a low oxygen mixture.Catheterization of the pulmonary artery was accomplished in the usual way (24,25). The position of the catheter was adjusted so that the tip lay just beyond the pulmonic valve. In those subjects in whom the wedged pressure was also measured, a special doublelumen catheter was used which allowed the tip to be wedged while the proximal lumen opened into the main pulmonary artery. With the catheter in place, a cannula was introduced into...