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Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)
 
 
 
 
 

Construction of a recombinant adenovirus containing the denV gene from bacteriophage T4 which can partially restore the DNA repair deficiency in xeroderma pigmentosum fibroblasts.

The denV gene from bacteriophage T4 encodes a pyrimidine dimer-specific endonuclease that has the capacity to initiate excision repair of DNA. Cells from excision repair-deficient xeroderma pigmentosum (XP) patients are able to carry out excision repair initiated by the denV gene product and introduction of the denV gene into XP cells results in the partial restoration of colony-forming ability after irradiation with UV light. In this work we have constructed a helper-independent recombinant human adenovirus, Ad5denV, which contains the denV gene. A 1.9 kb cartridge consisting of the denV gene flanked by the long terminal repeat (LTR) promoter from Rous sarcoma virus (RSV) and the simian virus 40 (SV40) polyadenylation (poly A) splice signals, was inserted into the E3 region of an E3 deletion mutant (Ad5d1E3) of adenovirus type 5. Infection of human fibroblasts and other permissive human cells with Ad5denV resulted in lytic infection and expression of the denV gene was confirmed by primer extension of infected cell RNA. The ability of the denV gene to restore the DNA repair deficiency in XP fibroblasts was examined using host cell reactivation of viral structural antigen formation for UV-irradiated adenovirus. The control virus, Ad5VSV, was also a recombinant which contained the gene for vesicular stomatitis virus glycoprotein G inserted into the E3 region of Ad5d1E3. UV survival of Ad5denV was similar to that of Ad5VSV following infection of two normal fibroblast strains and a Cockayne syndrome fibroblast strain, CS7SE, from complementation group B. In contrast, UV survival of Ad5denV was significantly greater than that for Ad5VSV after infection of three unrelated XP fibroblast strains from complementation groups A, C and E. However, UV survival of Ad5denV in the XP fibroblasts did not reach levels obtained in normal fibroblasts, indicating that restoration of the XP defect was partial.[1]

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