DNA topoisomerases are ubiquitous enzymes that are found in both prokaryotes and eukaryotes. Mechanistically, they cause DNA strands to pass through broken DNA-protein bridges in a concerted breakage and rejoining reaction in the DNA backbone. Although the precise function of many of these enzymes is not clear, their participation in replication, transcription, recombination and transposition has been suggested. This proposal is designed to investigate the role in replication and recombination. T4 DNA topoisomerase (a type II enzyme) consists of three non-identical subunits which are the products of T4 genes 39, 52 and 60. We propose to use recombinant DNA technology to clone these genes into high expression vectors such that individual components can be isolated and purified. The purified subunits will be examined individually and in combination, in order to determine if they can carry out any or part of the topoisomerization reactions characteristic of the complete enzyme. The site specific interactions of the T4 topoisomerase as well as its component subunits with DNA origins of replication will be examined using its ability to protect the target sites against digestion by DNaseI as assays. The genes for all three subunits, will be sequenced. Furthermore, its role in an excision event will be evaluated through the development of an in vitro system capable of resolving a duplication in the origin region of pBR322 DNA, using either the purified 39-protein alone or in conjunction with other cellular components as suggested by the in vivo process.
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