Creation and Repair of Specific DNA Double-Strand Breaks in Vivo Following Infection with Adenovirus Vectors Expressing Saccharomyces cerevisiae HO Endonuclease

Abstract

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.

In situ Detection of Specific DNA Double Strand Breaks using Rolling Circle Amplification

“…Coinfection of 293 cells with both viruses results in cleavage of the HO site to steady state levels of some 10 to 30% of the total viral genomes (data not shown), similar to that observed with the original AdE1::HO site virus. 13 Approximately 18 hours after infection of 293 cells under low m.o.i. condition, RCA was performed directly on 8-well culture slides.…”

“…Introduction of yeast HO endonuclease and its recognition site into cultured mammalian cells makes it possible to induce sequencespecific DNA DSBs. 13 The unique identification of RCA signals in cultures doubly infected with viruses carrying endonuclease and cleavage sites, and the nuclear localization of these signals, suggests that the sites of RCA were indeed sites of DSBs induced by this system. We also demonstrated that in situ RCA and immunofluorescence…”

“…The construction of virus Ad E1::HO Gene has been described in Nicolás et al 13 Virus Ad E1::HO Site B was constructed by first inserting the 127 nt BamHI to SmaI fragment, containing the MATa HO site from pWJ421, into the BamHI and EcoRV sites of the adenovirus shuttle vector pACE. 14 Virus was produced by cotransfecting 293 cells with the modified vector DNA and the adenovirus genome-containing plasmid pJM17.…”

“…Following plaque-purification, the recombinant structure of the genome was confirmed by restriction digestion and DNA sequence analysis of viral DNA. The HO site is oriented in the opposite direction to that in the Ad E1::HO Site virus described earlier, 13 with the oligonucleotide with ends complementary to 30-nt contiguous sequence within the HO site is then added. Annealing of this oligonucleotide to the denatured HO site will result in formation of a noncovalently closed circle that is next sealed with DNA ligase.…”

Adenovirus early E4 genes in viral oncogenesis

“…Functional analyses show that ORF6, although not ORF3, can inhibit V(D)J-joining (Boyer et al, 1999), a process that requires DNA PK. Moreover, Young and coworkers showed that both ORF3 and 6 can inhibit the repair of speci®c double-strand breaks induced by the Saccharomyces cerevisiae HO endonuclease in co-infected cells (Nicola s et al, 2000).…”