Mutations in two cysteine-histidine-rich clusters in adenovirus type 2 DNA polymerase affect DNA binding.
Several point and linker insertion mutations in two Cys-His-rich regions of adenovirus (Ad) DNA polymerase (Pol) gene have been expressed in recombinant vaccinia virus. The resulting mutant enzymes were analyzed in vitro for their effects on DNA synthesis activity, on Ad-specific initiation assays, on gel shifts of Ad origin sequences, and on interactions with adenovirus preterminal protein (pTP) and nuclear factor I (NFI). In general, mutants in downstream Cys-His sequences had a pronounced effect in these assays. Mutants in the upstream Cys-His region had a moderate effect on DNA synthesis and elongation but failed to make dCMP-pTP initiation complexes and failed to make specific shifted complexes in a gel retardation assay. These mutants could still bind to pTP and NFI in a coimmunoprecipitation experiment, suggesting that this upstream Cys-His region of Ad Pol is involved either in specific Ad DNA origin binding or in nonspecific DNA binding. Changing residues within Cys doublets in the downstream Cys-His region had pronounced effects on many Ad Pol functions such as DNA synthesis, DNA binding, and in vitro initiation; however, these mutants showed little reduction in binding to pTP and NFI; mutants at other cysteines or histidines within this region of Ad Pol did not appear to have an effect on enzyme function. This observation suggests that the downstream Cys-His region of Ad Pol is important for DNA binding and might fold into a Zn finger motif.
Joung
I
I
Department of Biochemistry, School of Medicine, University of Alabama, Birmingham 35294-0005.
Engler
J A
JA
D000256:Adenoviridae / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D022762:CCAAT-Enhancer-Binding Proteins; D002460:Cell Line; D002522:Chlorocebus aethiops; D003545:Cysteine / Q000235:genetics*; D004261:DNA Replication; D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000235:genetics* / Q000378:metabolism; D006367:HeLa Cells; D006639:Histidine / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D010957:Plasmids; D014157:Transcription Factors / Q000378:metabolism; D014407:Tumor Cells, Cultured; D051840:Y-Box-Binding Protein 1
D022762:CCAAT-Enhancer-Binding Proteins; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D014157:Transcription Factors; D051840:Y-Box-Binding Protein 1; C070856:YBX1 protein, human; D006639:Histidine; D004259:DNA-Directed DNA Polymerase; D003545:Cysteine