dc.contributor.advisor | Jones, Anita | en |
dc.contributor.advisor | Dryden, David | en |
dc.contributor.author | Ma, Long | en |
dc.date.accessioned | 2013-09-25T10:31:09Z | |
dc.date.available | 2013-09-25T10:31:09Z | |
dc.date.issued | 2012-11-28 | |
dc.identifier.uri | http://hdl.handle.net/1842/7836 | |
dc.description.abstract | 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. | en |
dc.contributor.sponsor | CSC (China Scholarship Council) | en |
dc.contributor.sponsor | Edinburgh University International MTEM Scholarship Scheme | en |
dc.contributor.sponsor | EaStChem | en |
dc.language.iso | en | |
dc.publisher | The University of Edinburgh | en |
dc.relation.hasversion | Ma, L.; Cockroft, S. L., Biological Nanopores for Single-Molecule Biophysics. ChemBioChem 2010, 11 (1), 25-34. | en |
dc.relation.hasversion | Wilson, C. P.; Boglio, C.; Ma, L.; Cockroft, S. L.; Webb, S. J., Palladium(II)-Mediated Assembly of Biotinylated Ion Channels. Chemistry-A European Journal 2011, 17 (12), 3465-3473. | en |
dc.relation.hasversion | Sabir, T.; Toulmin, A.; Ma, L.; Jones, A. C.; McGlynn, P.; Schroder, G. F.; Magennis, S. W., Branchpoint Expansion in a Fully Complementary Three-Way DNA Junction. J. Am. Chem. Soc. 2012, 134, 6280-6285. | en |
dc.subject | DNA conformation | en |
dc.subject | enzyme DNA systems | en |
dc.subject | fluorescence technique | en |
dc.title | Investigation of DNA conformation and enzyme-DNA systems using fluorescence techniques | en |
dc.type | Thesis or Dissertation | en |
dc.type.qualificationlevel | Doctoral | en |
dc.type.qualificationname | PhD Doctor of Philosophy | en |