Recombination and integration in Bacillus subtilis transformation: Involvement of DNA synthesis

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Using a transformable thymine-requiring strain of Bacillus subtilis as a recipient, it has been shown that there is no gross abnormality of DNA synthesis during the acquisition of competence and that transformation can occur during a time when the average amount of DNA synthesis in the recipient culture was no more than 0·15% of the normal cell cycle. A competent culture of SB566 ( try 2 thy ) was transferred to a medium containing 5-bromouracil at the time of addition of donor DNA. When the resulting lysate, following termination of transformation, was fractionated pycnographically, donor and recombinant activity banded predominantly in the light, unreplicated region. This shows that no significant amount of DNA synthesis is necessary for the initial stages of integration. Even if cells are first incubated in 5-bromouracil for some time before adding donor DNA, integrated donor activity is found only in the unreplicated position. Growth in 5-bromouracil and transfer to a heavy (D 2 O- 15 N) medium after transformation show that there is a lag in the replication of donor DNA. This lag is also reflected in the resistance of transformed cells to thymineless death and to the lethal effects of extensive incorporation of 5-bromouracil. Growth of a competent culture in 5-bromouracil immediately following transformation shows that when donor DNA replicates, it initially appears in a density region between light and hybrid. Such material most probably includes the transition point formed at the time of reinitiation of DNA synthesis in the transformed cell. On the basis of these results, a model is proposed in which donor DNA is integrated at the stationary replicating point of the competent cell, which is in a state of suspended DNA synthesis. The implications of this model are discussed in detail. An extension of the model to recombination in higher organisms is suggested, on the assumption that homologous chromosomes replicate synchronously through a common replicating point during the meiotic division.