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

Tautomerization is a fundamental process in chemistry and biology, where it plays a major role in vision and enzymatic reactions. Usually, extensive spectroscopic ensemble studies are required to identify a tautomeric equilibrium. For instance, indirect evidence for the fast motion of the two inner hydrogen atoms between the nitrogen atoms has been deduced for porphycene, 1 a constitutional isomer of porphyrin, from complex NMR 1,2 and fluorescence spectroscopy 3,4 studies in a solid host for both ground and excited singlet states.A fascinating aspect of single-molecule spectroscopy (SMS) is its capability to detect a chemical reaction in a single chromophore. This has been reported, for instance, for electron transfer, 5 conformational changes, 6 or photochromism. 7 These studies recorded variations in fluorescence decay curves, fluorescence intensity trajectories, or fluorescence spectra. The fast exchange of the hydrogens in porphyrin-like molecules, however, leads to a dynamic equilibrium of chemically equivalent structures and hence cannot be inferred from lifetime or spectral measurements. On the other hand, the two structures differ in the orientation of the transition dipole moments of the S 0 -S 1 transition. As determined from bulk fluorescence anisotropy studies, 4 the S 0 -S 1 transition moments in the two trans forms enclose an angle of about 70-80°( Figure 1A). We will exploit this information in the interpretation of the intensity patterns of fluorescence from single porphycene molecules.Single-molecule transition dipole moments have been used previously to probe the electric field distribution in the focal spot of a confocal microscope. [8][9][10] In this report, we show that the analysis of single-molecule fluorescence patterns in real space excited by an azimuthally polarized laser beam ( Figure 1B) provides an important alternative tool for analyzing structural changes in the large class of compounds where fluorescence resonance energy transfer (FRET), fluorescence decay curves, or fluorescence spectra cannot be used. Porphyrins and porphycenes are important representatives of such a reaction. We show for the first time the direct visualization of the two tautomers resulting from double hydrogen transfer in a single molecule of porphycene. Samples of porphycene immobilized in a 10-20-nm-thick PMMA layer have been prepared by spin coating of a 10 -9 M solution in toluene onto a quartz cover slide. Two-dimensional (2D) spatial images of the fluorescence intensities of individual molecules were obtained by raster scanning the sample in 20-nm steps per 5 ms. The fluorescence intensity is proportional to |p‚E| 2 , the square of the projection of the electric field E on the transition dipole moment p. Hence, the observed patterns reveal the orientations of p 8,9 and can be compared with numerical simulations taking into account the experimental parameters of our microscope.We observed a total of 66 spatially separated single-molecule patterns on one sample that revealed characteristic blinking and ...