Abstract
Human flap endonuclease 1 (FEN1) and related structure-specific 5'nucleases precisely identify and incise aberrant DNA structures during replication, repair and recombination to avoid genomic instability. Yet, it is unclear how the 5'nuclease mechanisms of DNA distortion and protein ordering robustly mediate efficient and accurate substrate recognition and catalytic selectivity. Here, single-molecule sub-millisecond and millisecond analyses of FEN1 reveal a protein-DNA induced-fit mechanism that efficiently verifies substrate and suppresses off-target cleavage. FEN1 sculpts DNA with diffusion-limited kinetics to test DNA substrate. This DNA distortion mutually 'locks' protein and DNA conformation and enables substrate verification with extreme precision. Strikingly, FEN1 never misses cleavage of its cognate substrate while blocking probable formation of catalytically competent interactions with noncognate substrates and fostering their pre-incision dissociation. These findings establish FEN1 has practically perfect precision and that separate control of induced-fit substrate recognition sets up the catalytic selectivity of the nuclease active site for genome stability.
Highlights
Biologically-critical, structure-specific 5’nucleases are highly conserved endo- or exo-nucleases that hydrolyze phosphodiester bonds that are one nucleotide into the 5’end of single-stranded(ss)/double-stranded(ds)-DNA junctions (Figure 1A), including nicks, gaps, flaps, bubbles and four-way junctions (Balakrishnan and Bambara, 2013; Finger et al, 2012; Tsutakawa et al, 2014; Tsutakawa and Tainer, 2012)
A major question in DNA damage recognition is whether the DNA distortion observed in proteinÀDNA complexes occurs spontaneously and is captured by the protein or if the protein actively sculpts the DNA into the distorted conformation (Figure 1C)
To determine which is the case for flap endonuclease 1 (FEN1), we started by establishing the conformational state of DF substrates alone using an ideal non-equilibrated (NonEQ) DF substrate containing 6 nt ssDNA 5’flap and 1 nt ssDNA 3’flap primers with no complementarity with the template strand (NonEQ DF-6,1)
Summary
Biologically-critical, structure-specific 5’nucleases are highly conserved endo- or exo-nucleases that hydrolyze phosphodiester bonds that are one nucleotide into the 5’end of single-stranded(ss)/double-stranded(ds)-DNA junctions (Figure 1A), including nicks, gaps, flaps, bubbles and four-way junctions (Balakrishnan and Bambara, 2013; Finger et al, 2012; Tsutakawa et al, 2014; Tsutakawa and Tainer, 2012) This conserved cleavage site despite diverse structures operates by uniformly binding to a bent junction to place the scissile phosphate near the active site (Liu et al, 2015; Orans et al, 2011; Tsutakawa et al, 2011) (Figure 1B).
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