Single-molecule studies of mycobacterial topoisomerase ia C-terminal domains and DNA

Abstract

Modern biochemical studies have revealed, and continue to reveal, many insights to protein-nucleic acid interactions. However, a weakness of bulk biochemical assays is the imparted restriction to beforeand- after type results. This causes in an obfuscation of individual protein behaviors and detailed time-dependent quantities such as kinetic rates. The ability to manipulate individual molecules and record data in real time is a large advantage of many single-molecule methods. Magnetic tweezers are ideal for studying DNA and the proteins which interact with it because they don't make physical contact with the sample and don't apply heat or free radicals. The work in this dissertation largely focuses on type IA topoisomerases and DNA. Type IA topoisomerases are present in all kingdoms of life and their core responsibility is simplifying topological complications of DNA during cellular processes. The current accepted model for how this simplification occurs is based on the topoisomerase cutting one strand of a locally denatured duplex and passing the intact strand through the opening. The topoisomerase studied in this dissertation, Mycobacterium smegmatis topoisomerase IA, is a common model for the type IA topoisomerase of the disease causing Mycobacterium tuberculosis. This protein has unique C-terminal domains which have been shown to be necessary for completion of its catalytic cycle. Part of this dissertation examines the functions of the C-terminal domains withe single-molecule magnetic tweezers by observing the relaxation activity of several protein variants which have had portions of their C-terminal domains removed. Given the prevalence of DNA use in biochemical experiments, it is desirable to remove any unknowns regarding its behavior prior to running an assay. One condition explored in this dissertation is how salt changes the DNA mechanical properties which govern its distribution of topological strain. This compounds with the knowledge that different organisms contain different distributions of salts and that may have a biological impact when studying proteins outside of their native conditions. To that end, this dissertation includes experiments and analyses which examine how different combinations and concentrations of salts impact DNA torsional responses.