Evidence of dynamics and disorder using NMR-spectroscopic techniques applied to human Flap-Endonuclease-1

Flap endonuclease 1 (FEN1) is a member of a 5’ nuclease superfamily involved in DNA replication and repair. FEN1 hydrolyses the phosphodiester bond one nucleotide into the duplex region of bifurcated double-flapped structures as found in lagging-strand DNA synthesis. These flap structures need to be cut in a very specific manner on the order of around 106 times per cell cycle. Therefore FEN1 is seen as an essential enzyme that maintains genomic integrity across all life forms. How FEN1 achieves its molecular recognition for a chemically very similar but structurally different DNA substrate and how it achieves catalysis on a biochemically relevant timescale are key questions to understand the protein system. This thesis describes some of the mechanistic studies used to understand how the structure and dynamics of hFEN1 relate to its function. It was proposed that T5 bacteriophage FEN was a catalytically perfect, or diffusion-limited enzyme, yet its main rate-limiting step after substrate binding was non-chemistry related. To ascertain whether this was true for hFEN1, the effect of leaving group pKa using 2’ modified double flapped substrates on rates of catalysis was measured. It was found that both apparent second order rates and first order single turnover rates of catalysis were insensitive to leaving group pKa. Furthermore by supplementing the reaction with glycerol, an unexpectedly high viscosity dependence was observed. The explanation for this is likely the presence of another physical step in the catalytic cycle affected by viscosity. Previous structural and biophysical studies of hFEN1 identified a helical arch, which was thought to be disordered as it could accommodate bulky 5’ flaps through it. Furthermore, the arch is key for positioning the 5’ flap into the active site. Using NMR spectroscopic techniques the solution state conformation of apo-hFEN1 was analysed. The arch was found to be disordered, but the C- terminal portion of it was transiently sampling α-helical φ,ψ space, while the other half was in an extended conformation. Another DNA recognition region, the α2-α3 loop was also found to be disordered. Various ligands and substrates were found to alter the structure and the dynamics of hFEN1. Addition of substrate DNA slowed the motion of the arch and α2-α3 loop to a millisecond timescale. Equally addition of a single monophosphate nucleotide had an effect on the dynamics of the top of the arch, despite binding in the active site. Furthermore, titration of calcium ions into the active site when DNA was present on the enzyme resulted in large perturbations to substrate recognition sites distant from the active site. This potentially links the specificity of these regions to activity within the active site.