Abstract
Flap endonuclease-1 (FEN-1) is a critical enzyme for DNA replication and repair. Intensive studies have been carried out on its structure-specific nuclease activities and biological functions in yeast cells. However, its specific interactions with DNA substrates as an initial step of catalysis are not defined. An understanding of the ability of FEN-1 to recognize and bind a flap DNA substrate is critical for the elucidation of its molecular mechanism and for the explanation of possible pathological consequences resulting from its failure to bind DNA. Using human FEN-1 in this study, we identified two positively charged amino acid residues, Arg-47 and Arg-70 in human FEN-1, as candidates responsible for substrate binding. Mutation of the Arg-70 significantly reduced flap endonuclease activity and eliminated exonuclease activity. Mutation or protonation of Arg-47 shifted cleavage sites with flap substrate and significantly reduced the exonuclease activity. We revealed that these alterations are due to the defects in DNA-protein interactions. Although the effect of the single Arg-47 mutation on binding activities is not as severe as R70A, its double mutation with Asp-181 had a synergistic effect. Furthermore the possible interaction sites of these positively charged residues with DNA substrates were discussed based on FEN-1 cleavage patterns using different substrates. Finally data were provided to indicate that the observed negative effects of a high concentration of Mg(2+) on enzymatic activity are probably due to the competition between the arginine residues and metal ions with DNA substrate since mutants were found to be less tolerant.
Highlights
Flap endonuclease-1 (FEN-1)1 proteins possess a flap endonuclease activity as well as a 5Ј to 3Ј exonuclease activity [1,2,3,4,5]
We have recently demonstrated that stimulation of eukaryotic FEN-1 activities by proliferating cell nuclear antigen (PCNA) is independent of its in vitro interaction via a consensus PCNA binding region [25], the interaction may be crucial for PCNA to recruit FEN-1 onto Okazaki fragment processing and DNA damage sites (24, 26 – 28)
To identify amino acid residues directly involved in substrate binding, we constructed a three-dimensional molecular model for human FEN-1 based on the available homologous crystal structures (29 –33)
Summary
Arg-47, Arg-70 Leu-340, Asp-341, Phe-343, Phe-344 Lys-354, Arg-355, Lys-356, Lys-365, Lys-366, Lys-367. To identify amino acid residues directly involved in substrate binding, we constructed a three-dimensional molecular model for human FEN-1 based on the available homologous crystal structures (29 –33). We have identified 10 amino acid residues including Arg-29, Arg, Arg-70, Arg-73, Lys-80, Lys-93, Lys-99, Arg-100, Arg-103, and Arg-104 as candidate residues that directly interact with DNA substrates. These residues are conserved in eukaryotes and are located on the surface of the molecule. Arg-70 might interact with the upstream doublestranded region of the DNA substrates, and Arg-47, possibly interacting with the upstream template region of DNA substrate, was revealed to play a role in determining cleavage sites via mutational analysis, biochemical assays, protonation, and competition experiments
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