Abstract
Human flap endonuclease-1 (hFEN1) catalyzes the divalent metal ion-dependent removal of single-stranded DNA protrusions known as flaps during DNA replication and repair. Substrate selectivity involves passage of the 5′-terminus/flap through the arch and recognition of a single nucleotide 3′-flap by the α2–α3 loop. Using NMR spectroscopy, we show that the solution conformation of free and DNA-bound hFEN1 are consistent with crystal structures; however, parts of the arch region and α2–α3 loop are disordered without substrate. Disorder within the arch explains how 5′-flaps can pass under it. NMR and single-molecule FRET data show a shift in the conformational ensemble in the arch and loop region upon addition of DNA. Furthermore, the addition of divalent metal ions to the active site of the hFEN1–DNA substrate complex demonstrates that active site changes are propagated via DNA-mediated allostery to regions key to substrate differentiation. The hFEN1–DNA complex also shows evidence of millisecond timescale motions in the arch region that may be required for DNA to enter the active site. Thus, hFEN1 regional conformational flexibility spanning a range of dynamic timescales is crucial to reach the catalytically relevant ensemble.
Highlights
Flap endonuclease 1 (FEN1) is a member of the 5′-nuclease superfamily that is predominantly involved in Okazaki fragment maturation, and has roles in long-patch base excision repair and ribonucleotide excision repair [1,2]
As we were most interested in the dynamics associated with catalysis, the human FEN1 (hFEN1)– 336 construct from X-ray crystallographic studies was used for NMR studies [11,21]
Expanded views of the shaded region can be found in Supplementary Figure S4. (B) Front and (C) rear views of the hFEN1 structure (3Q8K) [11] with labeled secondary structure elements and colored backbone to denote assigned or unassigned residues. (D) Relative peak height obtained from the 1H–15N-TROSY spectrum of hFEN1 plotted versus residue number show that loops are generally more intense and are lanked by decreasing peak intensities and sometimes missing residues
Summary
Flap endonuclease 1 (FEN1) is a member of the 5′-nuclease superfamily that is predominantly involved in Okazaki fragment maturation, and has roles in long-patch base excision repair and ribonucleotide excision repair [1,2]. All three of these pathways create bifurcated nucleic acid structures known as 5′-laps that must be removed precisely to create single-stranded (ss) and nicked double-stranded (ds) DNA products (Figure 1A). Human FEN1 (hFEN1) has been postulated to be a potential cancer therapeutic target [5,6], and evidence suggests that combinatorial targeting of hFEN1 has therapeutic relevance [7]. Speciicity is paramount as unwanted hydrolysis of DNA or RNA can be deleterious; how hFEN1 and paralogues achieve substrate and scissile phosphate diester speciicity has been an area of considerable debate
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