The origin binding protein of herpes simplex virus 1 binds cooperatively to the viral origin of replication oris.
The origin binding protein (OBP) or herpes simplex virus 1 has been expressed in Escherichia coli and used to study the role of multiple OBP binding sites in the herpes simplex virus #1 origin of replication, oris. Our results showed that the sequence CGTTCGCACTT was required for the binding of OBP to duplex DNA with high affinity. The minimal oris contains three repeats of this sequence or close derivatives thereof. Filter binding experiments have demonstrated that specific binding occurs to two of these repeats, box I and box II. An investigation using the DNase I footprinting technique revealed that the binding of OBP to box I and box II was cooperative and led to the formation of a highly organized complex in which the entire oris sequence was induced. We observed furthermore that the AT-rich sequence of the oris dyad was readily accessible to macromolecules even in the OBP.oris complex. The DNase I cleavage pattern of this sequence was, however, altered radically, indicating that a significant conformational change had occurred. A tentative structural model for the OBP-oris interaction is discussed on the basis of these observations.
Elias
P
P
Department of Medical Biochemistry, University of Göteborg, Sweden.
Gustafsson
C M
CM
Hammarsten
O
O
D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D003001:Cloning, Molecular; D004261:DNA Replication; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D018139:Simplexvirus / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D014764:Viral Proteins; C055789:origin-binding proteins, viral