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 express ed 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 infecte d cells, Both recombinant and endogenous I3 protein demonstrate a stri king affinity for single-stranded, but not for double-stranded, DNA. T he interaction with DNA is resistant to salt, exhibits low cooperativi ty, and appears to involve a binding site of approximately 10 nucleoti des, Electrophoretic mobility shift assays indicate that numerous I3 m olecules can bind to a template, reflecting the stoichiometric interac tion of I3 with DNA. Sequence analysis reveals that a pattern of aroma tic and charged amino acids common to many replicative single-stranded DNA binding proteins (SSBs) is conserved in I3, The inability to isol ate viable virus containing an interrupted I3 allele provides strong e vidence 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.