Recombination-dependent DNA replication stimulated by double-strand breaks in bacteriophage T4.

We analyzed the mechanism of recombination-dependent DNA replication in bacteriophage T4-infected Escherichia coli using plasmids that have sequence homology to the infecting phage chromosome. Consistent with prior studies, a pBR322 plasmid, initially resident in the infected host cell, does not replicate following infection by T4. However, the resident plasmid can be induced to replicate when an integrated copy of pBR322 vector is present in the phage chromosome. As expected for recombination-dependent DNA replication, the induced replication of pBR322 required the phage-encoded UvsY protein. Therefore, recombination-dependent plasmid replication requires homology between the plasmid and phage genomes but does not depend on the presence of any particular T4 DNA sequence on the test plasmid. We next asked whether T4 recombination-dependent DNA replication can be triggered by a double-strand break (dsb). For these experiments, we generated a novel phage strain that cleaves its own genome within the nonessential frd gene by means of the I-TevI endonuclease (encoded within the intron of the wild-type td gene). The dsb within the phage chromosome substantially increased the replication of plasmids that carry T4 inserts homologous to the region of the dsb (the plasmids are not themselves cleaved by the endonuclease). The dsb stimulated replication when the plasmid was homologous to either or both sides of the break but did not stimulate the replication of plasmids with homology to distant regions of the phage chromosome. As expected for recombination-dependent replication, plasmid replication triggered by dsbs was dependent on T4-encoded recombination proteins. These results confirm two important predictions of the model for T4-encoded recombination-dependent DNA replication proposed by Gisela Mosig (p. 120-130, in C. K. Mathews, E. M. Kutter, G. Mosig, and P. B. Berget (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.

Integration of plasmids into the bacteriophage T4 genome.

We have analyzed the integration of plasmids into the bacteriophage T4 genome via homologous recombination. As judged by genetic selection for a plasmid-borne marker, a mutation in phage gene uvsX or uvsY essentially blocked the integration of a plasmid with homology to the T4 genome but no phage replication origin (non-origin plasmid). The strict requirement for these two proteins suggests that plasmid integration can proceed via a strand-invasion reaction similar to that catalyzed in vitro by the T4-encoded strand-exchange protein (UvsX) in concert with UvsY and gp32. In contrast to the results with the non-origin plasmid, a mutation in uvsX or uvsY reduced the integration of a T4 replication origin-containing plasmid by only 3-10-fold. These results suggest that the origin-containing plasmid integrates by both the UvsXY-dependent pathway used by the non-origin plasmid and by a UvsXY-independent pathway. The origin-containing plasmid integrated into the phage genome during a uvsX- or uvsY-mutant infection of a recA-mutant host, and therefore origin-dependent integration can occur in the absence of both phage- and host-encoded strand-exchange proteins (UvsX and RecA, respectively).