Study on the interaction of DNA and a novel DNA repair promoting protein from the radioresistant bacterium

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In an effort to unravel the interaction of DNA and a DNA repair promoting protein PprA from the radioresistant bacterium Deinococcus radiodurans, DNA binding deficient mutant proteins were generated and biochemically analyzed. First, the pprA gene was mutagenized by random mutagenesis using hydroxylamine or error-prone PCR, and E. coli transformants that carries a mutated pprA gene were screened by the correlation of colony size and DNA-binding ability as a guide. Second, mutant PprA proteins were subjected to agarose gel retardation assay to check their DNA binding ability. As a result, total 24 mutant proteins were obtained. Mutation sites were clustered between nucleotide positions 133 and 216 in the middle region of the gene. This region plays an important role in DNA binding ability of PprA protein. Agarose gel retardation assay revealed that wild-type protein interacts with DNA in multimeric form. By using gel filtration it was estimated that PprA protein exists in a dodecamer complex in aqueous solution. On the other hand, the molecular weight of mutants obtained was smaller than that of the wild type, suggesting the correlation of protein association state and DNA binding ability. More elaborate studies using the other approaches, such as electron microscope imaging and crystal oscillator microbalance, are required to analyze the interaction mode of DNA and PprA protein. Information concerning mutation sites obtained in the present study will provide an important clue to clarify the stereostructure-activity relationship of PprA protein.