Topoisomerase III-alpha in Double Holliday Junction Dissolution

Abstract

<p>Topoisomerase III&alpha; (Top3&alpha;) is an essential component of the double Holliday junction (dHJ) dissolvasome complex in metazoans. Previous work has shown that Top3&alpha; and Bloom's helicase (Blm) are able to convergently migrate the dHJ to create solely non-crossover products, thus preserving genomic integrity. However, many questions remain about the details of this process. Using a combination of biochemical and genetic tools, including dHJ substrate assays, gel electrophoresis, EMSA, pulldowns, fly crosses, and electron microscopy, this work expands our knowledge of the dissolution reaction. Tail mutants of Top3&alpha; were created and tested in a series of <italic>in vitro</italic> assays. Through these experiments, I discovered that the C-terminus of Top3&alpha; is important for binding Blm, interacting with DNA, conveying RPA stimulation, and <italic>in vivo</italic> functionality. I also observed that dissolution is an extremely processive reaction, with no accumulation of intermediates prior to product formation. When a non-specific topoisomerase was used (Top1, a type IB), accumulation of an intermediate was evident; however, contrary to predicted models, direct observation revealed that this intermediate is not a hemicatenane structure and still requires branch migration. Modifications were also made to the dHJ substrate creation method so that multiple types of HJ substrates could be produced efficiently.</p>