The methodology we have developed for the identification of protein-DNA covalent bond will be applied to a number of other cases, including DNA gyrase, lambda integrase, phi X gene A enzyme, and calf thymus DNA topoisomerase. The phi X gene A enzyme is particularly interesting in that its sequence is known from the gene A nucleotide sequence, thus it is likely that we can identify the active site of the enzyme as well as the nature of the covalent linkage. Experiments will be designed to test the possibility of ATP-driven movement of DNA relative to an enzyme. The systems we will study include DNA gyrase, the type I restriction enzyme Eco R.K. and E. coli rep ATPase. Work on RNA polymerase will continue. Circular DNA containing only one kind of promoter will be constructed in vitro to carry out detailed studies of the unwinding of different promoter sequences by RNA polymerase.