The herpes simplex virus type 1 origin binding protein. Specific recognition of phosphates and methyl groups defines the interacting surface for a monomeric DNA binding domain in the major groove of DNA.
The UL9 gene of herpes simplex virus type 1 (HSV-1) encodes an origin binding protein (OBP). It is an ATP-dependent DNA helicase and a sequence-specific DNA-binding protein. The latter function is carried out by the C-terminal domain of OBP (DeltaOBP). We have now performed a quantitative analysis of the interaction between DeltaOBP and its recognition sequence, GTTCGCAC, in oriS. Initially optimal conditions for binding were carefully determined. We observed that complexes with different electrophoretic mobilities were formed. A cross-linking experiment demonstrated that nonspecific complexes containing 2 or more protein monomers per DNA molecule were formed at high protein concentrations. The specific complex formed at low concentrations of DeltaOBP had an electrophoretic mobility corresponding to a 1:1 complex. We then demonstrated that the methyl groups of thymine in the major groove were essential for high affinity binding. Changes in the minor groove had considerably smaller effects. Ethylation interference experiments indicated that specific contacts were made between OBP and three phosphates in the recognition sequence. Finally, these observations were used to present a model of the surface of DNA that interacts with DeltaOBP in a sequence-specific manner.
Simonsson
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S
Department of Medical Biochemistry, Göteborg University, Box 440, S. E. 405 30 Göteborg, Sweden.
Samuelsson
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Elias
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P
D001483:Base Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004265:DNA Helicases / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D004139:Dinitrofluorobenzene / Q000031:analogs & derivatives; D018259:Herpesvirus 1, Human / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D018741:Replication Origin; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism*
D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D014764:Viral Proteins; C064565:UL9 protein, Human herpesvirus 1; D004247:DNA; D004139:Dinitrofluorobenzene; D004265:DNA Helicases; C002041:1,5-difluoro-2,4-dinitrobenzene