Purification and characterization of UL9, the herpes simplex virus type 1 origin-binding protein.
UL9, the origin-binding protein of herpes simplex virus type 1 (HSV-1), has been overexpressed in an insect cell overexpression system and purified to homogeneity. In this report, we confirm and extend recent findings on the physical properties, enzymatic activities, and binding properties of UL9. We demonstrate that UL9 exists primarily as a homodimer in solution and that these dimers associate to form a complex nucleoprotein structure when bound to the HSV origin of replication. We also show that UL9 is an ATP-dependent helicase, capable of unwinding partially duplex DNA in a sequence-independent manner. Although the helicase activity of UL9 is demonstrable on short duplex substrates in the absence of single-stranded DNA-binding proteins, the HSV single-stranded DNA-binding protein ICP8 (but not heterologous binding proteins) stimulates UL9 to unwind long DNA sequences of over 500 bases. We were not able to demonstrate unwinding of fully duplex DNA sequences containing the HSV origin of replication. However, in experiments designed to detect origin-dependent unwinding, we did find that UL9 wraps supercoiled DNA independent of sequence or ATP hydrolysis.
Fierer
D S
DS
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892.
Challberg
M D
MD
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004265:DNA Helicases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D009036:Moths; D009698:Nucleoproteins / Q000378:metabolism; D018139:Simplexvirus / Q000201:enzymology* / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D009698:Nucleoproteins; D014764:Viral Proteins; C064565:UL9 protein, Human herpesvirus 1; D004265:DNA Helicases