Because of the intertwining nature of DNA molecules and the organization of DNA into loops and domains, topological problems arise inevitably as a result of DNA activities. The studies of this class of enzymes designed for the resolution of DNA are of fundamental importance. This proposal is designed to continue our efforts in the investigation of the structure- function and expression of type II ATP-dependent DNA topoisomerase using the enzymes encoded by the T even bacteriophages as the principle subject of investigations. Since type II DNA topoisomerases from all the diverse sources are apparently related evolutionary and structurally, principles learned from the T-phage system will be of general interest. They represent one of the simplest structural arrangements supported by well characterized genetics and biochemical tools since these genes have been cloned, sequenced, overexpressed and the subunits purified both as individual components as well as reconstituted enzyme complexes. The T4 enzyme consists of subunits defined by three genes, 39, 52, and 60, whereas the T2 enzyme consists of subunits defined by two genes, 39 and 60. We propose to dissect the functional domains of the T-phage genes by standard protein biochemistry as well as expressing the peptide domains independently to verify their role in the complex series of reactions relating to the ATP-utilization, cutting-rejoining and subunit interactions as part of the complete catalytic topoisomerase reaction. Ultimately, we will examine the fine structure of these proteins at the atomic level by X- ray crystallographic analyses. Since some anti-tumor drugs have as their targets type II DNA topoisomerase, including T-phage enzymes, drug resistance will also be used as a structural probe to identify mutations in DNA topoisomerase which may effect their function. Because of the structural conservation of all type II enzymes the drug-inhibition studies will provide valuable information toward the rational design of future drugs of this class. The expression of one of the three T4 topoisomerase genes, gene 60, is synthesized in a very unusual manner. A 50 nucleotide interruption is present in the coding region of the gene. The interruption in transcribed but it is not removed from the mRNA before translation. By site directed mutagenesis, and with the aid of beta-galactosidase reporter system, we propose to further define and test a model, based on alternate pairing of H-bound bases in the secondary structure of the mRNA, with which the translation of non-contiguous codons is accomplished.
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