The double helical structure of DNA mandates the existence of a mechanism for unwinding the helix to expose single-stranded DNA for use as a template by DNA polymerase. One mechanism provided is a class of enzymes, the helicases, that unwind DNA using chemical energy provided by NTP hydrolysis. Five helicases have been described in E. coli, helicases I, II, III, Rep protein and DnaB protein; the exact role of each in DNA metabolism-is just beginning to be elucidated. The long-range goal of this research program is to understand, in enzymatic and molecular terms, the mechanism of action of each helicase, and to define their respective roles in DNA replication, repair, recombination and conjugation. Helicases are likely to be involved in all aspects of DNA metabolism necessitating the presence of several enzymes in E. coli with helicase activity. Insight into the role of a specific helicase has been gained by determination of DNA substrate requirements, determination of reaction mechanism and elucidation of protein- protein interactions. Genetic analyses complement this biochemical approach. Studies of DNA substrate requirements for helicases II and Rep protein have been undertaken. The effect of additional proteins such as E. coli SSB on the unwinding reaction will be evaluated. In addition, a systematic search for previously unknown proteins that stimulate each helicase reaction will be carried out. Identification of such proteins, and the genes encoding them, will provide additional biochemical and genetic tools for determining precise roles for each helicase. A new helicase, the 75-kDa helicase has been discovered. Biochemical analysis of the unwinding reaction catalyzed by this enzyme and cloning of the gene encoding this helicase will be accomplished. Structure-function studies of the uvrD gene, using site-directed mutagenesis, are proposed. Identification of the ATPase, helicase and DNA binding sites are short-range goals. In summary, it is expected that these studies will enhance our understanding of complex reaction schemes such as excision repair, recombination and DNA replication. Knowledge of DNA substrate requirements for each helicase, the nature of protein-protein interactions between helicases and other proteins, and study of the relationship between helicases are required to understand the role of each helicase in DNA metabolism.
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