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

TitleCreation and repair of specific DNA double-strand breaks in vivo following infection with adenovirus vectors expressing Saccharomyces cerevisiae HO endonuclease.
Publication TypeJournal Article
Year of Publication2000
AuthorsNicolás AL, Munz PL, Falck-Pedersen E, Young CS
JournalVirology
Volume266
Issue1
Pagination211-24
Date Published2000 Jan 05
ISSN0042-6822
KeywordsAdenoviridae, Adenovirus E4 Proteins, Animals, Cell Line, Cloning, Molecular, Deoxyribonucleases, Type II Site-Specific, DNA, DNA Damage, DNA Repair, Genetic Vectors, Humans, Polymerase Chain Reaction, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Tumor Cells, Cultured
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.

DOI10.1006/viro.1999.0062
Alternate JournalVirology
PubMed ID10612676
Grant ListCA13696 / CA / NCI NIH HHS / United States
GM31452 / GM / NIGMS NIH HHS / United States
HL59312 / HL / NHLBI NIH HHS / United States

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