Studies on the site-specific self cleavage of G-quadruplexes and the topological properties of DNA

• Main Author: Hiew, Shu Hui
• Other Authors: School of Physical and Mathematical Sciences
• Format: Theses and Dissertations
• Language: English
• Published: 2012
• Subjects: DRNTU::Science
• Online Access: http://hdl.handle.net/10356/47844
• Institution: Nanyang Technological University

In this thesis, the studies are classified under two main themes, namely the studies on G-quadruplexes and studies on the topological properties of DNA. Firstly, the effects of several factors that could affect the self-cleaving reactions of G-quadruplex were examined in this study. The optimum self-cleaving reaction condition observed was between pH 7.4 – 7.6 with potassium ion stabilizing the G-quadruplex structure in the central cavity. It was found that Mg2+ is the best metal ion to catalyze the hydrolysis self-cleavage process, whereas Ca2+ catalyzes the self-cleaving reactions best when the ligand, methionine was introduced. Secondly, structural polymorphism and the effect of the structure of the resulting of G-quadruplexes with varying loop length was studied by designing series of oligonucleotides with four repeat guanines sequences, d(G4Tn)3G4 (where n = 1-6). It was found that as the loop length increases, the possibility of forming a loop increases, and the formation of the types of loops ascends: no loop < lateral loop < diagonal loop < external loop. The conformation of G-quadruplexes can be predicted if we know the T-loop length. Lastly, the topological properties of DNA was explored. In order to examine if DNA curvature had any effect on human topoisomerase I, a series of DNA possessing intrinsic curvature was design, and it was found that hTopo I recognizes the curvature and binds preferably to the curved DNA with a higher degree of curvature. On the other hand, besides using EcTopo I, DNA gyrase was also able to help determine the superhelical density of circular DNA. Some very interesting findings were made with regards to the decatenation of kDNA by human Topoisomerase II alpha, whereby the enzyme might possibly be able to introduce supercoiling into circular DNA, when the self-crossing of the DNA backbone exist in the solution. Also, when the decatenation reaction was incubated past the completion of the decatenation, the enzyme seems to “re-catenate” the kDNA circles.