RECOMBINATION-DEPENDENT DNA-REPLICATION STIMULATED BY DOUBLE-STRAND BREAKS IN BACTERIOPHAGE-T4

We analyzed the mechanism of recombination-dependent DNA replication i n bacteriophage T4-infected Escherichia coil using plasmids that have sequence homology to the infecting phage chromosome. Consistent with p rior studies, a pBR322 plasmid, initially resident in the infected hos t cell, does not replicate following infection by T4. However, the res ident plasmid can be induced to replicate when an integrated copy of p BR322 vector is present in the phage chromosome. As expected for recom bination-dependent DNA replication, the induced replication of pBR322 required the phage-encoded UvsY protein, Therefore, recombination-depe ndent plasmid replication requires homology between the plasmid and ph age genomes but does not depend on the presence of any particular T4 D NA sequence on the test plasmid. We next asked whether T4 recombinatio n-dependent DNA replication can be triggered by a double-strand break (dsb). For these experiments, we generated a novel phage strain that c leaves its own genome within the nonessential frd gent! by means of th e I-TevI endonuclease (encoded within the intron of the, wild-type tri gene), The dsb within the phage chromosome substantially increased th e replication of plasmids that carry T4 inserts homologous to the regi on of the dsb (the plasmids are not themselves cleaved by the endonucl ease). The db stimulated replication when the plasmid was homologous t o either or both sides of the break but did not stimulate the replicat ion of plasmids with homology to distant regions of the phage chromoso me, As expected for recombination-dependent replication, plasmid repli cation triggered by dsbs was dependent on T4-encoded recombination pro teins, These results confirm two important predictions of the model fo r T4 recombination-dependent DNA replication proposed by Gisela Mosig (p. 120-130, in C. K. Mathews, E. M. Kutter, G. Mosig, and P. B. Berge t (ed.), Bacteriophage T4, 1983). In addition, replication stimulated by dsbs provides a site-specific version of the process, which should be very useful for mechanistic studies.