Characterization of the single-stranded DNA binding protein encoded by the vaccinia virus I3 gene.
The 34-kDa protein encoded by the I3 gene of vaccinia virus is expressed at early and intermediate times postinfection and is phosphorylated on serine residues. Recombinant I3 has been expressed in Escherichia coli and purified to near homogeneity, as has the protein from infected cells. Both recombinant and endogenous I3 protein demonstrate a striking affinity for single-stranded, but not for double-stranded, DNA. The interaction with DNA is resistant to salt, exhibits low cooperativity, and appears to involve a binding site of approximately 10 nucleotides. Electrophoretic mobility shift assays indicate that numerous I3 molecules can bind to a template, reflecting the stoichiometric interaction of I3 with DNA. Sequence analysis reveals that a pattern of aromatic and charged amino acids common to many replicative single-stranded DNA binding proteins (SSBs) is conserved in I3. The inability to isolate viable virus containing an interrupted I3 allele provides strong evidence that the I3 protein plays an essential role in the viral life cycle. A likely role for I3 as an SSB involved in DNA replication and/or repair is discussed.
Rochester
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SC
Department of Cell Biology, Cornell University Medical College, New York, New York 10021, USA.
Traktman
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P
D000595:Amino Acid Sequence; D017104:Bacteriophage M13 / Q000235:genetics; D002460:Cell Line; D002845:Chromatography; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D005814:Genes, Viral; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016296:Mutagenesis; D009841:Oligonucleotides / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D014616:Vaccinia virus / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C111416:I3 protein, Vaccinia virus; D009841:Oligonucleotides; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D012965:Sodium Chloride; D012694:Serine