The 3′-Flap Pocket of Human Flap Endonuclease 1 Is Critical for Substrate Binding and Catalysis

Abstract

Flap endonuclease 1 (FEN1) proteins, which are present in all kingdoms of life, catalyze the sequence-independent hydrolysis of the bifurcated nucleic acid intermediates formed during DNA replication and repair. How FEN1s have evolved to preferentially cleave flap structures is of great interest especially in light of studies wherein mice carrying a catalytically deficient FEN1 were predisposed to cancer. Structural studies of FEN1s from phage to human have shown that, although they share similar folds, the FEN1s of higher organisms contain a 3-extrahelical nucleotide (3-flap) binding pocket. When presented with 5-flap substrates having a 3-flap, archaeal and eukaryotic FEN1s display enhanced reaction rates and cleavage site specificity. To investigate the role of this interaction, a kinetic study of human FEN1 (hFEN1) employing well defined DNA substrates was conducted. The presence of a 3-flap on substrates reduced K m and increased multiple-and single turnover rates of endonucleolytic hydrolysis at near physiological salt concentrations. Exonucleolytic and fork-gap-endonucleolytic reactions were also stimulated by the presence of a 3-flap, and the absence of a 3-flap from a 5-flap substrate was more detrimental to hFEN1 activity than removal of the 5-flap or introduction of a hairpin into the 5-flap structure. hFEN1 reactions were predominantly rate-limited by product release regardless of the presence or absence of a 3-flap. Furthermore, the identity of the stable enzyme product species was deduced from inhibition studies to be the 5-phosphorylated product. Together the results indicate that the presence of a 3-flap is the critical feature for efficient hFEN1 substrate recognition and catalysis.In eukaryotic DNA replication and repair, various bifurcated nucleic acid structure intermediates are formed and must be processed by the appropriate nuclease. Two examples of biological processes that create bifurcated DNA intermediates are Okazaki fragment maturation (1, 2) and long patch excision repair (3). In both models, a polymerase executes strand-displacement synthesis to create a double-stranded DNA (dsDNA) 6 two-way junction from which a 5Ј-flap structure protrudes. The penultimate step of both pathways is the cleavage of this flap structure to create a nicked DNA that is then ligated. Because the bifurcated DNA structures that are formed in the aforementioned processes can theoretically occur anywhere in the genome, the nuclease associated with the cleavage of 5Ј-flap structures in eukaryotic cells, which is called flap endonuclease 1 (FEN1), must be capable of cleavage regardless of sequence. Therefore, FEN1 nucleases, which are found in all kingdoms of life (4), have evolved to recognize substrates based upon nucleic acid structure and strand polarity (5, 6).The Okazaki fragment maturation pathway of yeast has become a paradigm of eukaryotic lagging strand DNA synthesis. In the yeast model, bifurcated intermediates with large single-stranded DNA (ssDNA) 5Ј-flap structures are imprecisely cleaved by DNA2 ...

Flap Endonuclease Activity of Gene 6 Exonuclease of Bacteriophage T7

“…The helical arch region of hFEN1 is disordered in the absence of a DNA substrate but becomes structured when DNA binds to the protein (35,64). This disordered state of hFEN1 can thread a flap containing a short duplex and then cleave the flap, suggesting the ability to thread DNA containing 5Ј-flaps with adducts or DNA with long 5Ј-flaps (42,43,53). However, the structure of T5 exonuclease, whose structure is close to the gp6 model, shows that the helical arch is in the substrate-free state, suggesting that this conformational transition does not occur upon binding the DNA substrate.…”

Neutralizing Mutations of Carboxylates That Bind Metal 2 in T5 Flap Endonuclease Result in an Enzyme That Still Requires Two Metal Ions

“…These include enzymes that catalyze structure-specific reactions of an apparently disparate group of nucleic acid substrates including DNA bubbles (xeroderma pigmentosum complementation group G (XPG)) (35), four-way junctions (GEN-1 (xeroderma pigmentosum complementation group G-like gap endonuclease, a putative human Holliday junction resolvase)) (36), as well as flapped, nicked, and 3Ј-overhang DNAs (FEN and EXO1) (2,20,37,38). Because all of these enzymes carry out the same chemical transformation, like T5FEN, they too will require two ions to effect phosphate diester hydrolysis.…”

Repair complexes of FEN1 endonuclease, DNA, and Rad9-Hus1-Rad1 are distinguished from their PCNA counterparts by functionally important stability

“…The FEN1 substrates are in fact double-flap DNA (dfDNA) with DNA on the opposite side of the 5′ flap, forming a single nucleotide 3′ flap when bound to the enzyme (2,3). By removing the 5′ ssDNA or RNA flap from such substrates, FEN1 produces a single nicked product that could be sealed by the subsequent action of a DNA ligase (4). Consistent with its crucial role in DNA replication and repair, FEN1 is highly expressed in all proliferative tissues, and its activity is key for the maintenance of genomic integrity (5).…”