STUDIES OF BACTERIOPHAGE MU DNA TRANSPOSITION & EXCISION

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We propose to investigate certain specific aspects of DNA transposition and excision in bacteriophage Mu. We would like to follow up our surprising observation that transposition of Mu can occur via two pathways and that one of the factors that influence the choice of the pathways could be the proportion of the two proteins A and B that are required for transposition. We will exploit this observation to study specifically one or the other pathway. Besides its involvement in DNA transposition, the A protein also brings about the excision of Mu DNA sequences from their integrated sites. We have cloned the A gene under the strong lacUV5 promoter. Upon overproduction of A, we can detect in vivo, linear excised Mu DNA. We propose to use this observation to test whether excised Mu DNA is an intermediate in transposition. We also plan to reproduce the excision reaction in vitro and thus have an assay to purify the A protein. Eventually we plan to obtain pure transposition proteins in sufficient amounts to study the physico-chemical interactions between the proteins and their substrates.