SUMMARY Flap endonuclease (FEN1), essential for DNA replication and repair, removes RNA and DNA 5′-flaps. FEN1 5′ nuclease superfamily members acting in nucleotide excision repair (XPG), mismatch repair (EXO1) and homologous recombination (GEN1) paradoxically incise structurally distinct bubbles, ends or Holliday junctions, respectively. Here structural and functional analyses of human FEN1:DNA complexes show structure-specific, sequence-independent recognition for nicked dsDNA bent 100° with unpaired 3′- and 5′-flaps. Above the active site, a helical cap over a gateway formed by two helices enforces ssDNA threading and specificity for free 5′-ends. Crystallographic analyses of product and substrate complexes reveal that dsDNA binding and bending, the ssDNA gateway, and double-base unpairing flanking the scissile phosphate control precise flap incision by the two-metal-ion active site. Superfamily conserved motifs bind and open dsDNA, direct the target region into the helical gateway permitting only non-base-paired oligonucleotides active site access, and support a unified understanding of superfamily substrate specificity.

The collapse of stalled replication forks is a major driver of genomic instability. Several committed mechanisms exist to resolve replication stress. These pathways are particularly pertinent at telomeres. Cancer cells that use Alternative Lengthening of Telomeres (ALT) display heightened levels of telomere-specific replication stress, and co-opt stalled replication forks as substrates for break-induced telomere synthesis. FANCM is a DNA translocase that can form independent functional interactions with the BLM-TOP3A-RMI (BTR) complex and the Fanconi anemia (FA) core complex. Here, we demonstrate that FANCM depletion provokes ALT activity, evident by increased break-induced telomere synthesis, and the induction of ALT biomarkers. FANCM-mediated attenuation of ALT requires its inherent DNA translocase activity and interaction with the BTR complex, but does not require the FA core complex, indicative of FANCM functioning to restrain excessive ALT activity by ameliorating replication stress at telomeres. Synthetic inhibition of FANCM-BTR complex formation is selectively toxic to ALT cancer cells.

Protein nucleases and RNA enzymes depend on divalent metal ions to catalyze the rapid hydrolysis of phosphate diester linkages of nucleic acids during DNA replication, DNA repair, RNA processing, and RNA degradation. These enzymes are widely proposed to catalyze phosphate diester hydrolysis using a "two-metal-ion mechanism." Yet, analyses of flap endonuclease (FEN) family members, which occur in all domains of life and act in DNA replication and repair, exemplify controversies regarding the classical two-metal-ion mechanism for phosphate diester hydrolysis. Whereas substrate-free structures of FENs identify two active site metal ions, their typical separation of >4 Å appears incompatible with this mechanism. To clarify the roles played by FEN metal ions, we report here a detailed evaluation of the magnesium ion response of T5FEN. Kinetic investigations reveal that overall the T5FEN-catalyzed reaction requires at least three magnesium ions, implying that an additional metal ion is bound. The presence of at least two ions bound with differing affinity is required to catalyze phosphate diester hydrolysis. Analysis of the inhibition of reactions by calcium ions is consistent with a requirement for two viable cofactors (Mg 2؉ or Mn 2؉). The apparent substrate association constant is maximized by binding two magnesium ions. This may reflect a metal-dependent unpairing of duplex substrate required to position the scissile phosphate in contact with metal ion(s). The combined results suggest that T5FEN primarily uses a two-metal-ion mechanism for chemical catalysis, but that its overall metallobiochemistry is more complex and requires three ions.Key cellular processes such as DNA replication, DNA repair, RNA processing, and RNA degradation require the rapid hydrolysis of the phosphate diester linkages of nucleic acids. The uncatalyzed hydrolysis of phosphate diesters under biological conditions is an extremely slow process with an estimated half-life of 30 million years at 25°C (1). Protein nucleases and RNA enzymes produce rate enhancements of 10 15 -10 17 to allow this reaction to proceed on a biologically useful time scale. Most enzymes catalyzing phosphate diester bond hydrolysis have a requirement for divalent metal ions. Based largely upon crystallographic observations, most metallonucleases are proposed to catalyze reactions using a two-metal-ion mechanism (Fig. 1a) analogous to that suggested for the phosphate monoesterase alkaline phosphatase (2, 3), although this view is not universally accepted. Three recent reviews present contrasting views on the roles of metal ions in protein nuclease and RNA enzyme reactions and illustrate this controversy (4 -6).One family of metallonucleases over which there has been considerable mechanistic debate are the flap endonucleases (FENs) 3 (7-12), which are present in all domains of life and play a key role in DNA replication and repair. Unlike most metallonucleases, which typically possess a cluster of three or four active site carboxylates, the FEN active site is constructed...

FENs (flap endonucleases) and related FEN-like enzymes [EXO-1 (exonuclease-1), GEN-1 (gap endonuclease 1) and XPG (xeroderma pigmentosum complementation group G)] are a family of bivalent-metal-ion-dependent nucleases that catalyse structure-specific hydrolysis of DNA duplex-containing nucleic acid structures during DNA replication, repair and recombination. In the case of FENs, the ability to catalyse reactions on a variety of substrates has been rationalized as a result of combined functional and structural studies. Analyses of FENs also exemplify controversies regarding the two-metal-ion mechanism. However, kinetic studies of T5FEN (bacteriophage T5 FEN) reveal that a two-metal-ion-like mechanism for chemical catalysis is plausible. Consideration of the metallobiochemistry and the positioning of substrate in metal-free structures has led to the proposal that the duplex termini of substrates are unpaired in the catalytically active form and that FENs and related enzymes may recognize breathing duplex termini within more complex structures. An outstanding issue in FEN catalysis is the role played by the intermediate (I) domain arch or clamp. It has been proposed that FENs thread the 5'-portion of their substrates through this arch, which is wide enough to accommodate single-stranded, but not double-stranded, DNA. However, FENs exhibit gap endonuclease activity acting upon substrates that have a region of 5'-duplex. Moreover, the action of other FEN family members such as GEN-1, proposed to target Holliday junctions without termini, appears incompatible with a threading mechanism. An alterative is that the I domain is used as a clamp. A future challenge is to clarify the role of this domain in FENs and related enzymes.