Early intermediates in bacteriophage T4 DNA replication and recombination.

We investigated, by density gradients and subsequent electron microscopy, vegetative T4 DNA after single or multiple infection of Escherichia coli with wild-type T4. Our results can be summarized as follows. (i) After single infection (i.e., when early intermolecular recombination could not occur), most, if not all, T4 DNA molecules initiated the first round of replication with a single loop. (ii) After multiple infection, recombinational intermediates containing label from both parents first appeared as early as 1 min after the onset of replication, long before all parental DNA molecules had finished their first round and before secondary replication was detectable. (iii) At the same time, in multiple infections only, complex, highly branched concatemeric T4 DNA first appeared. (iv) Molecules in which two loops or several branches were arranged in tandem were only found after multiple infections. (v) Secondary loops within primary loops were seen after both single and multiple infections, but they were rare and many appeared off center. Thus, recombination in wild-type T4-infected cells occurred very early, and the generation of multiple tandem loops or branches in vegetative T4 DNA depended on recombination. These results are consistent with the previous finding (A. Luder and G. Mosig, Proc. Natl. Acad. Sci. U.S.A. 79:1101-1105, 1982) that most secondary growing points of T4 are not initiated from origin sequences but from recombinational intermediates. By these and previous results, the various DNA molecules that we observed are most readily explained as intermediates in DNA replication and recombination according to a model proposed earlier to explain various other aspects of T4 DNA metabolism (Mosig et al., p. 277-295, in D. Ray, ed., The Initiation of DNA Replication, Academic Press, Inc., New York, 1981).