To study DNA double-strand break (DSB) repair in mammalian cells, the Saccharomyces cerevisiae HO endonuclease gene, or its recognition site, was cloned into the adenovirus E3 or E1 regions. Analysis of DNA from human A549 cells coinfected with the E3::HO gene and site viruses showed that HO endonuclease was active and that broken viral genomes were detectable 12 h postinfection, increasing with time up to approximately 30% of the available HO site genomes. Leftward fragments of approximately 30 kbp, which contain the packaging signal, but not rightward fragments of approximately 6 kbp, were incorporated into virions, suggesting that broken genomes were not held together tightly after cleavage. There was no evidence for DSB repair in E3::HO virus coinfections. In contrast, such evidence was obtained in E1::HO virus coinfections of nonpermissive cells, suggesting that adenovirus proteins expressed in the permissive E3::HO coinfection can inhibit mammalian DSB repair. To test the inhibitory role of E4 proteins, known to suppress genome concatemer formation late in infection (Weiden and Ginsberg, 1994), A549 cells were coinfected with E3::HO viruses lacking the E4 region. The results strongly suggest that the E4 protein(s) inhibits DSB repair.

The joining of DNA double-strand breaks in vivo is frequently accompanied by the loss of a few nucleotides at the junction between the interacting partners. In vitro systems mimic this loss and, on detailed analysis, have suggested two models for the mechanism of end-joining. One invokes the use of extensive homologous side-by-side alignment of the partners prior to joining, while the other proposes the use of small regions of homology located at or near the terminus of the interacting molecules. to discriminate between these two models, assays were conducted both in vitro and in vivo with specially designed substrates. In vitro, molecules with limited terminal homology were capable of joining, but analysis of the junctions suggested that the mechanism employed the limited homology available. In vivo, the substrates with no extensive homology end-joined with equal efficiency to those with extensive homology in two different topological arrangements. Taken together, these results suggest that extensive homology is not a prerequisite for efficient end-joining, but that small homologies close to the terminus are used preferentially, as predicted by the modified single-strand annealing model.

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We have studied the temporal and functional relationships between DNA replication and recombination in adenovirus-infected cells by using Southern blot hybridization to detect recombinant products among intracellular viral genomes. The data show that recombination can be detected soon after DNA replication has commenced and that the proportion of recombinant products increases thereafter. To determine the functional relationship between DNA replication and recombination, replication was blocked with the protein synthesis inhibitor anisomycin, the replication inhibitor cytosine arabinoside, and conditionally lethal mutations in either the virus-specified DNA-binding protein or the DNA polymerase. All treatments that directly or indirectly blocked DNA replication caused a delay in the appearance of recombinant products and a marked decline in their abundance relative to products of parental genotype. These data strongly suggest that DNA replication and recombination are interrelated, either because both processes share functions or because DNA structures produced by replication are suitable substrates for recombination. In addition, we have shown that some recombination function(s) is intrinsically thermolabile at 40.9°C, even in wild-type crosses, since the appearance of recombinant products is delayed and their extent is reduced compared with that from crosses performed at 39.9°C.

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