MECHANISM OF NUCLEIC ACID CLEAVAGE BY HYDROXYL RADICAL

羟基自由基是由电离辐射产生的，通过提取脱氧核糖中的氢原子引起DNA断裂。以非序列特异性方式切割DNA的能力使该试剂成为足迹实验中用于获得DNA， RNA及其与溶液中细胞蛋白复合物的结构信息的有效工具。尽管羟基自由基足迹技术被广泛使用，但关于羟基自由基切割核酸的机制的信息很少。用19bp DNA探针进行的初始氘同位素效应实验表明，脱氧核糖的5′和4′氢的溶剂可及性越大，与羟基自由基反应时氘同位素效应越大。本研究旨在扩展这些初始氘同位素动力学效应实验，并进一步研究羟基自由基裂解DNA的机制。RNA切割的机制也将用这种技术来研究。这些实验的结果将通过允许在脱氧核糖和核糖氢的水平上解释反应性而不是整个糖段来增加从足迹实验中获得的信息量。这些进展将有助于提供更多关于溶液中生物分子的结构细节。

Hydroxyl radical is produced by ionizing radiation and causes DNA scission by abstracting the hydrogen atoms of deoxyribose. The ability to cleave DNA in a non-sequence specific manner has made this agent an effective tool in footprinting experiments used to gain structural information about DNA, RNA, and their complexes with cellular proteins in solution. Despite the widespread use of the hydroxyl radical footprinting technique, very little information is available about the mechanism by which hydroxyl radical cleaves nucleic acids. Initial deuterium isotope effect experiments with a 19-bp DNA probe demonstrated that the greater solvent accessibility of the 5' and 4' hydrogens of the deoxyribose correlated with larger deuterium isotope effects upon reaction with hydroxyl radical. The proposed work seeks to expand these initial deuterium kinetic isotope effect experiments and investigate further the mechanism of DNA cleavage by hydroxyl radical. The mechanism of RNA cleavage will also be examined with this technique. The results of these experiments will increase the amount of information obtained from the footprinting experiments by allowing the reactivity to be interpreted at the level of the deoxyribose and ribose hydrogens instead of the entire sugar moiety. These advances will help to provide more structural details about biomolecules in solution.