Creation and repair of specific DNA double-strand breaks in vivo followinginfection with adenovirus vectors expressing Saccharomyces cerevisiae HO endonuclease

To study DNA double-strand break (DSB) repair in mammalian cells, the Sacch aromyces cerevisiae HO endonuclease gene, or its recognition site, was clon ed into the adenovirus E3 or E1 regions. Analysis of DNA from human A549 ce lls coinfected with the E3::HO gene and site viruses showed that HO endonuc lease was active and that broken viral genomes were detectable 12 h postinf ection, increasing with time up to similar to 30% of the available HO site genomes. Leftward fragments of similar to 30 kbp, which contain the packagi ng signal, but not rightward fragments of similar to 6 kbp, were incorporat ed into virions, suggesting that broken genomes were not held together tigh tly after cleavage. There was no evidence for DSB repair in E3::HO virus co infections. In contrast, such evidence was obtained in E1::HO virus coinfec tions 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 concateme r formation late in infection (Weiden and Ginsberg, 1994), A549 cells were coinfected with E3::HO viruses lacking the 54 region. The results strongly suggest that the 54 protein(s) inhibits DSB repair. (C) 2000 Academic Press .