Flap endonucleases (FENs) have essential roles in DNA processing. They catalyze exonucleolytic and structure-specific endonucleolytic DNA cleavage reactions. Divalent metal ions are essential cofactors in both reactions. The crystal structure of FEN shows that the protein has two conserved metal-binding sites. Mutations in site I caused complete loss of catalytic activity. Mutation of crucial aspartates in site II abolished exonuclease action, but caused enzymes to retain structure-specific (flap endonuclease) activity. Isothermal titration calorimetry revealed that site I has a 30-fold higher affinity for cofactor than site II. Structure-specific endonuclease activity requires binding of a single metal ion in the high-affinity site, whereas exonuclease activity requires that both the high- and low-affinity sites be occupied by divalent cofactor. The data suggest that a novel two-metal mechanism operates in the FEN-catalyzed exonucleolytic reaction. These results raise the possibility that local concentrations of free cofactor could influence the endo- or exonucleolytic pathway in vivo.

Previous structural studies on native T5 5 nuclease, a member of the flap endonuclease family of structure-specific nucleases, demonstrated that this enzyme possesses an unusual helical arch mounted on the enzyme's active site. Based on this structure, the protein's surface charge distribution, and biochemical analyses, a model of DNA binding was proposed in which single-stranded DNA threads through the archway. We investigated the kinetic and substrate-binding characteristics of wild-type and mutant nucleases in relation to the proposed model. Five basic residues R33, K215, K241, R172, and R216, are all implicated in binding branched DNA substrates. All these residues except R172 are involved in binding to duplex DNA carrying a 5 overhang. Replacement of either K215 or R216 with a neutral amino acid did not alter k cat appreciably. However, these mutant nucleases displayed significantly increased values for K d and Km. A comparison of flap endonuclease binding to pseudoY substrates and duplexes with a single-stranded 5 overhang suggests a better model for 5 nuclease-DNA binding. We propose a major revision to the binding model consistent with these biophysical data.T he flap endonucleases, or 5Ј nucleases, are structure-specific endonucleases that also possess 5Ј-3Ј exonucleolytic activity. They are involved in processing substrates with 5Ј singlestranded tails such as those that arise during nick translation and replication and in some DNA damage-repair pathways (1, 2). These enzymes bind substrates containing a single-stranded 5Ј end and a duplex region such as 5Ј overhangs (5OVHs), 5Ј flaps, and pseudoY (Ps-Y) substrates (Fig. 1;. It has been suggested that the single-stranded 5Ј tail of such substrates threads through the 5Ј nuclease (4). Several prokaryotic and archaeal flap endonuclease structures have been solved (6-10). Because none of the reported structures contained bound DNA substrates, the precise mode of nucleic acid binding is unclear. These nucleases share significant primary sequence conservation as well as extensive structural similarities (11)(12)(13)(14). A central ␤-sheet carrying many of the core metal-binding ligands is a striking feature of all 5Ј-nuclease structures reported to date. This core region contains acidic residues responsible for binding the two divalent metal ions required for nuclease activity in vitro. A series of helices and loops comprise the enzyme's core. The most variable region seems to be that comprising a helical arch in T5 5Ј nuclease, the observation of which led us to develop a model of substrate binding (ref. 8; Fig. 2). In T5 5Ј nuclease, the archway delineates a hole able to accommodate a threaded single-stranded nucleic acid. Although a similar hole is present in the Methanococcus jannaschii homologue (9), this region often appears disordered (6, 7) or as a folded loop in the structure of the Pyrococcus furiosus homologue (10). The conceptual model for flap endonuclease-DNA binding has been widely accepted (1,7,9,10,15,16). Nevertheless, this model suffers f...

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5'-3' exonucleases are essential for D N A replication, recombination and repair. T5 5' nuclease is a member of this group which exhibit 5'-3' exonuclease and structure-specific endonuclease activity. Our previous studies suggested that endo-and exonuclease activity are separable (1,2). Site-directed mutagenesis and biochemical analyses were used to investigate the role of absolutely conserved residues situated near the metal-binding sites in T5 5' nuclease. For example, mutations of a conserved tyrosine were found to increase the k, 4-45 fold (wild-type has k, of 10 nM) but retained full endo-and 5-15% exo-nuclease activity. Variation in the steady state kinetic parameters of wildtype and mutant T5 5' nuclease with p H demonstrated that protonation of Lys-83 is critical for DNA-binding (3). The results suggest that the endo-and exo-nucleolytic activities of the T5 enzyme are distinct and utilise different mechanisms to effect substrate cleavage.The 5' nucleases are required for both DNA replication and repair. T5 5' nuclease is a member of this group which exhibit 5'-3' exonuclease and structure-specific endonuclease activity. It is proposed that single-stranded DNA threads through a hole observed in the X-ray structure of such nucleases '.Structure-function studies suggest that different mechanisms for endo-and exonucleolytic hydrolysis are used by this enzyme*. Site-directed mutagenesis was employed to investigate the role of two acidic and two basic residues thought to be involved in cofactor and substrate binding respectively. These conserved residues form an integral part of a helix-loop-helix feature ', representing residues 189-224 of the T5 exonuclease sequence. Using quantitative band shift assays with different substrates and mutant proteins we determined the impact of mutation upon binding and catalysis. Our data lead us to propose an alternative DNA binding model for T5 exonuclease.Nucleic Acids Res., 25:4224-4229 382~90-93 29 Differential binding of Mn(I1) to bacteriophage T5 5' nuclease and vital role of metal site I1 in exonucleolytic cleavage 5' nucleases are essential for DNA replication and repair. They catalyze exonucleolytic hydrolysis of phosphodiester bonds in a variety of nucleic acid, also exhibit structure-specific endonucleolytic activity, allowing them to cleave bifucations of flap structures containing free 5'-end. Crystallographic and biochemical studies suggest single-stranded substrates become threaded through 5' nucleases, where hydrolysis is affected with the aid of divalent metal cations. Here we show the T5 5' nuclease to be promiscuous in metal ions as cofactor. By varying concentrations of various transition metals the cleavage pattern can be dramatically changed so that less than 1% of the products were caused by exonucleolytic cleavage. ITC data demenstrate differential binding of Mn(I1) to wild-type nuclease with total stochiometry of 2, while Co(I1) can occupy site I with stochiometry of 1 only. Mutagenesis of two aspatate prevents Mn(I1) from occupying metal site 11, thi...

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