Experiments-aimed at elucidating the biological role of Z-DNA have taken two forms. Natural sequences which can form Z-DNA have been identified and proteins have been purified which bind better to Z- DNA than B-DNA in vitro. However, the sequences have not been shown to form Z-DNA in vivo, nor have the proteins been shown to bind Z-DNA in vivo. We recently purified three proteins from coli which bind specifically to Z-DNA in vitro, prepared monoclonal antibodies against them, and cloned their genes. For the first time reagents are available that make possible molecular analyses of ,genes encoding Z-DNA binding proteins and extensive biochemical studies of these proteins. The goal of these studies is to elucidate the biological role of Z-DNA in E. coli by determining the function of these Z-DNA binding proteins. We will sequence their genes and locate them on the E. coli map, and analyze the migration of the proteins on 2-D PACE. Comparison of this information with available data banks will reveal if data on these proteins or genes has been collected. Using allelic replacement and constructs overexpressing or producing antisense mRNA for these genes we will generate null mutants or conditional lethals and strains which overexpress these genes ;or produce reduced levels of these proteins. We will examine the mutants for defects in processes executed at the DNA level: replication, recombination, repair, supercoiling, and transcription. If we obtain conditional lethals we will clone mutants in other genes which suppress the original mutant phenotype. Using highly purified protein from strains overexpressing these genes we will carry out an extensive study of the interaction of these proteins with Z-DNA. We will precisely quantitate the specificity of these proteins for Z-DNA. Chemical modification experiments will reveal details, at the base pair level, of the interaction. We will identify the in vivo binding sites of these proteins by immunoprecipitating UV cross- linked protein-DNA complexes from cell extracts and analyzing the precipitated DNA by cloning and sequencing. Purified proteins will also be assayed for NTPase, nuclease, topoisomerase, and recombination activities. Since so little is known about what Z- DNA does in a cell, we believe that E. coli, which is amenable to genetic analysis and is one of the simplest and best characterized laboratory organisms, is an ideal system in which to begin studying the function of Z-DNA. These studies will have far reaching implications since they will provide clues to the function of Z- DNA in organisms other than E. coli.
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