Investigation of DNA conformation and enzyme-DNA systems using fluorescence techniques
As a structural analogue of adenine (6-aminopurine), 2-aminopurine (2AP) is a powerful fluorescent probe, when substituted in DNA in place of the natural adenine. Time-resolved fluorescence measurements of 2AP-labeled oligonucleotides, together with steady-state spectroscopy give us an in-depth view of DNA-enzyme interactions, especially the conformational dynamics in solution phase. Herein, this technique has been extended to the study of the transient unzipping of DNA bases, to investigate the structure of three-way junction (3WJ), and the role of base unzipping in the mechanism of human flap endonuclease (FEN). Seven 2AP-labelled 3WJs were investigated, each containing only one 2AP base in place of adenine. In four of the 3WJs, 2AP was placed in the long duplex region of an arm; while in the other three 3WJs, 2AP was placed near or in the branch point. Comparative time-resolved fluorescence measurements on the 3WJs and corresponding ssDNA and dsDNA controls were made to study the base dynamics, in particular the possibility of unzipping in the vicinity of the branch point. In combination with single-molecule FRET measurements and molecular dynamics simulations, the local and global structure of a DNA 3WJ in solution could be unraveled. It was found to adopt a Y-shaped, pyramidal structure, in which the bases adjacent to the branch point are unzipped, despite the full Watson-Crick complementarity of the molecule. Human flap endonuclease (hFEN) is divalent metal ion-dependent phosphodiesterase. hFEN carries out structure-specific hydrolysis of 5’ bifurcated DNA endonucleolytically. Cleavage occurs at a position one nucleotide into the downstream duplex region. Previous structural, biochemical and modeling studies suggested a double-nucleotide unzipping mechanism at single/double strand junctions for scissile phosphate placement. To confirm this mechanism, 2AP time-resolved fluorescence spectroscopy was used to investigate nucleotide unzipping in hFEN substrates. 2AP was substituted at positions +1 and -1 (relative to the scissile phosphodiester) respectively, in double flap substrates. A series of hFEN mutants including Y40A, R100A, K93A, were used in this study. In the experiments, ssDNA, dsDNA substrates, DNA substrate-enzyme complexes were investigated in order to elucidate the enzyme-induced distortion of the substrate at the +1 and -1 positions. TseI is a thermophilic type II restriction enzyme which has ideal activity at an elevated temperature. It is able to recognise and cut the 5 bp palindromic sequence of 5’-GCWGC-3’ (W=A or T). A range of biophysical methods have been applied to investigate this enzyme, including size-exclusion chromatography; fluorescence anisotropy (Kd value determination); denaturing HPLC for DNA cleavage analysis on matched and mismatched substrates; fluorescence-based activity assay (KM, Vmax, kcat, specificity constant values determination); steady-state fluorescence measurements (DNA-enzyme interaction study). The DNA cleavage characteristics of TseI were fully studied and it was found that it cuts A:A and T:T mismatches in CAG and CTG repeats. This potentially makes it a useful tool for exploring unusual DNA structures containing super-long CAG and CTG repeats which are involved in the aetiology of some neurodegenerative diseases, such as Huntington’s disease (HD). EcoP15I is a type III restriction-modification enzyme whose recognition sequence is 5-CAGCAG-3’. Methyltransferase EcoP15I (M.EcoP15I) adds a methyl group to the second adenine, in the presence of cofactor S-adenosyl methionine (SAM). SDS-PAGE, densitometry and size-exclusion HPLC were applied to confirm that EcoP15I adopts a Res1Mod2 stoichiometry in solution. The large structural distortion of its substrate (base flipping) by M.EcoP15I was investigated by both steady-state and time-resolved fluorescence. Also, nine 120 mer DNA duplexes, each containing two reversely oriented recognition sites were used to study matched and mismatched sequence cleavage by R.EcoP15 and a cleavage pattern was revealed.