Exploring the structural features of DNA and the effect of DNA binders through a potpourri of biophysical techniques.

摘要

A number of biological roles have been proposed for quadruplex, also referred to as G4 or tetraplex, DNAs. These motifs are structurally different from the traditional duplex DNA, predominantly found in biological systems. The presence of quadruplex DNA may lead to errors in some biological processes and be required in others. Proteins that interact with quadruplex DNA have been identified including those that cause Bloom's and Werner's syndromes. There is compelling evidence that quadruplex structures might play a vital role in immunoglobulin switch regions, formation of the synaptonemal complexes during recombination and telomere maintenance.; Fluorescence dyes which selectively bind to quadruplex DNAs were identified. Some of these molecules inhibit telomerase, and are cytotoxic towards tumor cells indicating a role for quadruplex DNA in telomere function. A gel based method using selective staining, was developed to identify quadruplex structures amongst mixtures. The method was combined with 32P radiolabeled samples to study DNA sequences over a wide range of concentrations and salt conditions. This method was used to study the human telomere sequence among others.; While investigating the structural aspects of porphyrins binding to quadruplex DNA, we discovered the unique ability of these porphyrins to catalyze the conversion to parallel stranded structures. The porphyrins were able to convert any given quadruplex motif to a mixture of parallel stranded conformers. This property was investigated in detail using NMR, fluorescence spectroscopy, CD spectroscopy, stopped flow experiments and gel electrophoresis.; A new fluorescence based method was developed to investigate DNA bending and flexibility. The "equilibrium cyclization method" was used to study the A-tract and mismatched DNA sequences. The method was also used to study protein-DNA interactions. Studies HU binding to DNA reveals HU's preference for a flexible DNA. The study shows that HU bends the DNA upon binding and the binding constants obtained by the equilibrium cyclization method was in good agreement with those independently obtained by other methods. The technique was also used to monitor the formation of quadruplex structures and kinetics of DNA cutting by restriction nucleases.