Vaccinia DNA topoisomerase I promotes illegitimate recombination in Escherichia coli.

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RESUMO

Vaccinia virus encapsidates a Mr 32,000 type IDNA topoisomerase. Although the vaccinia gene encoding the topoisomerase is essential for virus growth, the role of the enzyme in vivo remains unclear. In the present study, the physiologic consequences of vaccinia topoisomerase action have been examined in a heterologous system, Escherichia coli. The vaccinia topoisomerase gene was inducibly expressed in an int-lambda lysogen BL21(DE3) using a T7 RNA polymerase-based transcription system. Expression of active topoisomerase in this context resulted in recA-dependent lysogenic induction as well as cell lysis. Surprisingly, topoisomerase expression also effected a 200-fold increase in the titer of infectious lambda phage, apparently by promoting int-independent prophage excision. This effect was not observed during lysogenic induction with nalidixic acid. Restriction analysis of genomic DNA from plaque-purified excisants revealed (in 10 of 10 cases) gross alterations of the DNA structure around the att site relative to the structure of the parental phage DE3. It is construed therefore that vaccinia DNA topoisomerase I acts to promote illegitimate recombination in E. coli.

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