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

In eukaryotes, DNA polymerase δ (Pol δ) bound to the proliferating cell nuclear antigen (PCNA) replicates the lagging strand and cooperates with flap endonuclease 1 (FEN1) to process the Okazaki fragments for their ligation. We present the high-resolution cryo-EM structure of the human processive Pol δ-DNA-PCNA complex in the absence and presence of FEN1. Pol δ is anchored to one of the three PCNA monomers through the C-terminal domain of the catalytic subunit. The catalytic core sits on top of PCNA in an open configuration while the regulatory subunits project laterally. This arrangement allows PCNA to thread and stabilize the DNA exiting the catalytic cleft and recruit FEN1 to one unoccupied monomer in a toolbelt fashion. Alternative holoenzyme conformations reveal important functional interactions that maintain PCNA orientation during synthesis. This work sheds light on the structural basis of Pol δ's activity in replicating the human genome.

When converging replication forks meet during replication termination, the CMG (Cdc45-MCM2-7-GINS) helicase is polyubiquitylated by CRL2 Lrr1 and unloaded from chromatin by the p97 ATPase. Here, we investigate the signal that triggers CMG unloading in Xenopus egg extracts using single-molecule and ensemble approaches. We show that converging CMGs pass each other and keep translocating at the same speed as before convergence, whereafter they are rapidly and independently unloaded. When CMG unloading is blocked, diverging CMGs do not support DNA synthesis, indicating that after bypass CMGs encounter the nascent lagging strands of the converging fork and then translocate along double-stranded DNA (dsDNA). However, translocation on dsDNA is not required for CMG's removal from chromatin because in the absence of nascent strand synthesis, converging CMGs are still unloaded. Moreover, recombinant CMG added to nuclear extract undergoes ubiquitylation and disassembly in the absence of any DNA, and DNA digestion triggers CMG ubiquitylation at stalled replication forks. Our findings suggest that DNA suppresses CMG ubiquitylation during elongation and that this suppression is relieved when CMGs converge, leading to CMG unloading.

Protein-induced fluorescence enhancement (PIFE) is a popular tool for characterizing protein-DNA interactions. PIFE has been explained by an increase in local viscosity due to the presence of the protein residues. This explanation, however, denies the opposite effect of fluorescence quenching. This work offers a perspective for understanding PIFE mechanism and reports the observation of a phenomenon that we name protein-induced fluorescence quenching (PIFQ), which exhibits an opposite effect to PIFE. A detailed characterization of these two fluorescence modulations reveals that the initial fluorescence state of the labeled mediator (DNA) determines whether this mediator-conjugated dye undergoes PIFE or PIFQ upon protein binding. This key role of the mediator DNA provides a protocol for the experimental design to obtain either PIFQ or PIFE, on-demand. This makes the arbitrary nature of the current experimental design obsolete, allowing for proper integration of both PIFE and PIFQ with existing bulk and single-molecule fluorescence techniques.