A 269-amino-acid segment with a pseudo-leucine zipper and a helix-turn-helix motif codes for the sequence-specific DNA-binding domain of herpes simplex virus type 1 origin-binding protein.
The UL9 gene of herpes simplex virus (HSV) codes for a DNA-binding protein (OBP) that interacts sequence specifically with the origin of replication. This protein is essential for HSV DNA replication in cultured cells. The UL9 gene was cloned into a plasmid vector downstream of the SP6 RNA polymerase promoter. By using in vitro transcription and translation systems, a full-length OBP was synthesized. This synthetic protein is recognized by an antiserum generated against the C-terminal decapeptide of OBP and is functionally active in binding to OriS sequence specifically. The in vitro-synthesized protein has sequence specificity for binding similar to that found for the in vivo-generated OBP. A total of 14 in-frame deletion and insertion mutants of the UL9 gene were generated and expressed in vitro. Using these deletion mutants, we determined that the 269-amino-acid stretch defined by amino acids 564 to 832 localizes the OriS-specific DNA-binding domain. The N-terminal boundary is between amino acids 565 and 596, while the C terminus lies between amino acids 833 and 805. This segment contains a helix-turn-helix moiety and a pseudo-leucine zipper, neither of which alone can support DNA binding. The other leucine zipper from amino acids 150 to 173 is not required for the in vitro sequence-specific DNA-binding activity of OBP.
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Department of Microbiology, University of Texas Health Science Center, San Antonio 78284-7758.
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D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D004261:DNA Replication; D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D015870:Gene Expression; D005814:Genes, Viral; D016350:Leucine Zippers / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011233:Precipitin Tests; D011487:Protein Conformation; D018139:Simplexvirus / Q000235:genetics* / Q000254:growth & development; D012995:Solubility; D014709:Vero Cells / Q000382:microbiology; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D014764:Viral Proteins; C055789:origin-binding proteins, viral; D000255:Adenosine Triphosphate; D012321:DNA-Directed RNA Polymerases