Abstract
The structure- and strand-specific phosphodiesterase flap endonuclease-1 (FEN1), the prototypical 5′-nuclease, catalyzes the essential removal of 5′-single-stranded flaps during replication and repair. FEN1 achieves this by selectively catalyzing hydrolysis one nucleotide into the duplex region of substrates, always targeting the 5′-strand. This specificity is proposed to arise by unpairing the 5′-end of duplex to permit the scissile phosphate diester to contact catalytic divalent metal ions. Providing the first direct evidence for this, we detected changes induced by human FEN1 (hFEN1) in the low-energy CD spectra and fluorescence lifetimes of 2-aminopurine in substrates and products that were indicative of unpairing. Divalent metal ions were essential for unpairing. However, although 5′-nuclease superfamily-conserved active-site residues K93 and R100 were required to produce unpaired product, they were not necessary to unpair substrates. Nevertheless, a unique arrangement of protein residues around the unpaired DNA was detected only with wild-type protein, suggesting a cooperative assembly of active-site residues that may be triggered by unpaired DNA. The general principles of FEN1 strand and reaction-site selection, which depend on the ability of juxtaposed divalent metal ions to unpair the end of duplex DNA, may also apply more widely to other structure- and strand-specific nucleases.
Highlights
Structure-specific phosphodiesterases play essential cellular roles by recognizing and acting on aberrant nucleic acid structures
The structure that is recognized by eukaryotic flap endonuclease-1 (FEN1) proteins is a two-way junction known as a 50-30double flap that has a 50-flap of any length and a single nucleotide 30-flap (Figure 1B)
When FEN1-catalyzed hydrolysis occurs one nucleotide into the 50-duplex, the resulting product is nicked DNA that can be immediately joined by DNA ligase
Summary
Structure-specific phosphodiesterases play essential cellular roles by recognizing and acting on aberrant nucleic acid structures. To return to the duplex state and thereby restore the genome, the ends of intact duplexes contained within these more complex structures must undergo siteselective strand-specific phosphate diester hydrolyses In line with this key role in maintaining genome integrity, defects in structure-sensing nucleases lead to a range of diseases, including cancer [1,2]. Other proteinsequence-related 50-nucleases include EXO1, the 50-nuclease that catalyzes resection of duplex, nicked and gapped DNAs during mismatch and double-strand break repair, and XPG, the 50-nuclease of nucleotide excision repair that targets DNA bubbles. Another family member, GEN1, processes DNA four-way (Holliday) junctions. Members of the 50-nuclease family act on a diverse range of substrates, one feature of their
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