Biochemical analysis of adenovirus type 5 DNA-binding protein mutants.
We previously reported the isolation and functional characterization of seven adenovirus type 5 (Ad5) DNA-binding protein (DBP) point mutants (Quinn, C. O., and Kitchingman, G. R. (1986) J. Virol. 60, 653-661). Six of the seven mutants were defective in their ability to help adeno-associated virus replicate its DNA. To determine the level at which the mutations affect this function of the DBP, we analyzed several properties of the mutant proteins. All are transported to the nucleus and are post-translationally phosphorylated to the same extent. The half-lives of the proteins, measured by pulse-chase, were nearly identical to that of the wild-type DBP. The mutant DBPs were examined for their ability to bind to single-stranded DNA (ssDNA). Mutations in amino acids 322, 323, and 470 lowered the affinity of the DBP for ssDNA, while a mutation in amino acid 181 had no affect. Combinations of mutations in amino acid 470 with either 322 or 323 did not further lower the affinity of the protein for ssDNA. These data indicate that the functional defect for adeno-associated virus helper activity of the six mutants is due mainly, if not totally, to their reduced affinity for single-stranded DNA. These experiments have thus identified a functional domain of the adenovirus type 5 DBP potentially involved in DNA-protein interactions. Comparisons with temperature-sensitive DBP mutants indicate that the conserved region mutants are functionally distinct and represent a new class of DBP mutants.
Neale
G A
GA
Department of Virology and Molecular Biology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101.
Kitchingman
G R
GR
D000256:Adenoviridae / Q000032:analysis* / Q000235:genetics; D000595:Amino Acid Sequence; D001692:Biological Transport; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006207:Half-Life; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D009154:Mutation; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D013329:Structure-Activity Relationship; D014162:Transfection; D014779:Virus Replication
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D010710:Phosphates