The self-association of guanine bases in a tetrameric square planar arrangement was first determined in the early 1960s. The tetramer, commonly termed the G-quartet, can stack upon other G-quartets to form four-stranded helices termed G-quadruplexes. Bioinformatics studies have revealed that guanine-rich sequences with the propensity to adopt these structures are found in telomeric DNA and throughout the human genome, particularly in gene promoter regions. It is thought that the location of these sequences is not a coincidence and that the folding potential of guanine-rich DNA in vivo may play an important role in biological events such as gene regulation. Repetitive guanine tracts of G-quadruplex-forming DNAs form highly polymorphic structures with parallel or antiparallel strand orientations, depending the on ionic condition and the length of the connecting loops, and can be assembled as inter- or intra-molecular complexes. While extensive research has demonstrated their formation in vitro, there is little direct evidence to support their formation in vivo. With the exception of the single-stranded telomeric DNA, all genomic guanine-rich sequences are always present in the duplex configuration. Therefore, these structures will need to compete with the duplex that is normally generated with the complementary cytosine-rich strand. In order for this to happen would first require the local dissociation of the strands. Negative supercoiling results from the unwinding of the DNA helix and is known to provide energy to facilitate the formation of a number of alternative DNA structures. The work described in this thesis therefore aims to investigate the formation of G-quadruplexes under negatively supercoiled conditions. This was examined by preparing plasmids that contained multiple copies of G-rich oligonucleotides, based on the sequences (G3T)n and (G3T4)n, cloned into the pUC19 vector. The formation of G-quadruplexes within these repeats has been assessed using the chemical probes dimethyl sulphate (DMS) and potassium permanganate, and the single-strand specific endonuclease S1. DMS probing revealed some evidence for G-quadruplex formation in (G3T)n sequences, though this was not affected by DNA supercoiling. However, probing with KMnO4 failed to detect exposed thymines in the loop regions, though there was some supercoil-dependent reactivity in the surrounding sequences, suggesting that this had been affected by the G-rich region. In contrast, the (G3T4)n sequences did not demonstrate protection from DMS, suggesting that G-quadruplex formation had not taken place. Surprisingly, the KMnO4 reactions identified structural alterations around, but not within, the inserted G-rich fragments. S1 nuclease digestions did not detect any structural perturbations in any of the sequences apart from a mutant plasmid containing an inverted quadruplex repeat at the 3’-end. Two-dimensional gel electrophoresis of DNA topoisomers was also conducted to detect any supercoil-dependent B-DNA to quadruplex transitions. Neither the (G3T)n nor (G3T4)n plasmids showed any such structural changes. However, the mutant plasmid did demonstrate some supercoil-dependent changes, though these may correspond to cruciform rather than G-quadruplex formation. These results do not support the suggestion that negative supercoiling can induce the formation of G-quadruplex structures.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:628666 |
| Date | January 2013 |
| Creators | Sekibo, Doreen |
| Contributors | Fox, Keith |
| Publisher | University of Southampton |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://eprints.soton.ac.uk/367077/ |
Page generated in 0.0019 seconds