Multiple functions of the adenovirus DNA-binding protein are required for efficient viral DNA synthesis.
Mutational analysis within the amino-terminal (N-t) domain of the adenovirus DNA-binding protein (DBP) defined a region (aa 2-38) important for DBP function. Several viruses carrying lesions in this region of DBP showed reduced accumulation of viral DNA and infectious virions. Characterization of one of these mutants, H5in800, indicated that the N-t domain affects viral DNA synthesis in vivo. The reduction in DNA synthesis was not due to a change in the amount or nuclear location of the H5in800 DBP. Expression of other early genes in H5in800-infected cells was similar to that seen in wild-type Ad5-infected cells, suggesting that the depression of DNA synthesis was not due to disruption of DBP's role in early gene expression. The H5in800 and wild-type DBP also had comparable affinities for single-stranded DNA and functioned with similar efficiencies in two DNA elongation assays. Prior studies have shown that the carboxyl-terminal (C-t) domain of DBP was responsible for these two activities. Together these results suggest that DBP has at least two separable functions in viral DNA replication in vivo and that both domains of the protein are necessary for full activity. The intragenic complementation between the N-t mutant H5in800 and the C-t mutant H5in804 supports this model.
Brough
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DE
Waksman Institute, Rutgers, State University of New Jersey, Piscataway 08855-0759.
Droguett
G
G
Horwitz
M S
MS
Klessig
D F
DF
D000260:Adenoviruses, Human / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D002460:Cell Line; D004261:DNA Replication; D004277:DNA, Single-Stranded; D004279:DNA, Viral / Q000096:biosynthesis* / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015967:Gene Expression Regulation, Viral; D005816:Genetic Complementation Test; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D009154:Mutation; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D014764:Viral Proteins