Positioning the 5′-flap junction in the active site controls the rate of flap endonuclease-1–catalyzed DNA cleavage

Abstract

Flap endonucleases catalyze cleavage of single-stranded DNA flaps formed during replication, repair, and recombination and are therefore essential for genome processing and stability. Recent crystal structures of DNA-bound human flap endonuclease (hFEN1) offer new insights into how conformational changes in the DNA and hFEN1 may facilitate the reaction mechanism. For example, previous biochemical studies of DNA conformation performed under non-catalytic conditions with Ca have suggested that base unpairing at the 5'-flap:template junction is an important step in the reaction, but the new structural data suggest otherwise. To clarify the role of DNA changes in the kinetic mechanism, we measured a series of transient steps, from substrate binding to product release, during the hFEN1-catalyzed reaction in the presence of Mg We found that whereas hFEN1 binds and bends DNA at a fast, diffusion-limited rate, much slower Mg-dependent conformational changes in DNA around the active site are subsequently necessary and rate-limiting for 5'-flap cleavage. These changes are reported overall by fluorescence of 2-aminopurine at the 5'-flap:template junction, indicating that local DNA distortion ( disruption of base stacking observed in structures), associated with positioning the 5'-flap scissile phosphodiester bond in the hFEN1 active site, controls catalysis. hFEN1 residues with distinct roles in the catalytic mechanism, including those binding metal ions (Asp-34 and Asp-181), steering the 5'-flap through the active site and binding the scissile phosphate (Lys-93 and Arg-100), and stacking against the base 5' to the scissile phosphate (Tyr-40), all contribute to these rate-limiting conformational changes, ensuring efficient and specific cleavage of 5'-flaps.

Implementing fluorescence enhancement, quenching, and FRET for investigating flap endonuclease 1 enzymatic reaction at the single-molecule level

“…3 D & 3E) [72] ; this is consistent with findings from bulk cleavage assays [75] , [90] , [91] . Hence, the 5′ flap release is instantaneous [55] , [72] , [91] , the single turnover rate for catalysis ( k STO ) can be determined from the inverse of the average dwell time prior to cleavage ( k STO = 1/Avg τ bending-Flap ). This k STO includes DNA bending, protein ordering for active site assembly, chemistry and 5′ flap release.…”

“…The probability density plot of the distributions of the dwell time before cleavage ( τ bending-Flap ) of NonEQ and EQ DF-6,1 showed that both substrates are cleaved with similar kinetics ( Fig. 3 D & 3E) [72] ; this is consistent with findings from bulk cleavage assays [75] , [90] , [91] . Hence, the 5′ flap release is instantaneous [55] , [72] , [91] , the single turnover rate for catalysis ( k STO ) can be determined from the inverse of the average dwell time prior to cleavage ( k STO = 1/Avg τ bending-Flap ).…”

Structure of the processive human Pol δ holoenzyme

“…However, handing off a bent DNA is not a must for FEN1 activity since FEN1 can actively bend the nick junction in diffusion-limited kinetics 54,55 . Nick translation occurs at rates that are 10-fold faster than nick DNA product release by FEN1 16,[55][56][57] . This suggests that Pol δ and FEN1 must be actively handing off their products during nick translation.…”

Resolution of the Holliday junction recombination intermediate by human GEN1 at the single-molecule level

“…This distorted structure projects the HJ for the second GEN1 monomer to bind before licensing catalysis. DNA-induced fit binding has also been illustrated in FEN1 and Exo1, two members of the 5′ nuclease superfamily to which GEN1 belongs (39,57). Increasing structural evidence also suggests that the proteins undergo conformational changes upon DNA binding, which control active-site assembly in GEN1 (35,36), FEN1 (37,41) and Exo1 (42,43).…”

“…In the case of FEN1, the protein uses an induced-fit mechanism where it actively pulls out a nucleotide at the 3′ end of the nick junction to drive protein ordering (39,40). This step locks the protein and the DNA interactions to verify their ability to promote the transition state (39,57,58).…”