Functional properties of the herpes simplex virus type I origin-binding protein are controlled by precise interactions with the activated form of the origin of DNA replication.
The herpes simplex virus, type I origin-binding protein, OBP, is a superfamily II DNA helicase encoded by the UL9 gene. OBP binds in a sequence-specific and cooperative way to the viral origin of replication oriS. OBP may unwind partially and introduce a hairpin into the double-stranded origin of replication. The formation of the novel conformation referred to as oriS* also requires the single-stranded DNA-binding protein, ICP8, and ATP hydrolysis. OBP forms a stable complex with oriS*. The hairpin in oriS* provides a site for sequence-specific attachment, and a single-stranded region triggers ATP hydrolysis. Here we use Escherichia coli exonuclease I to map the binding of the C-terminal domain of OBP to the hairpin and the helicase domains to the single-stranded tail. The helicase domains cover a stretch of 23 nucleotides of single-stranded DNA. Using streptavidin-coated magnetic beads, we show that OBP may bind two copies of double-stranded DNA (one biotin-labeled and the other one radioactively labeled) but only one copy of oriS*. It is the length of the single-stranded tail that determines the stoichiometry of OBP.DNA complexes. OBP interacts with the bases of the single-stranded tail, and ATP hydrolysis is triggered by position-specific interactions between OBP and bases in the single-stranded tail of oriS*.
Macao
Bertil
B
Department of Medical Biochemistry, Göteborg University, Box 440, S-405 30 Göteborg, Sweden.
Olsson
Monica
M
Elias
Per
P
D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D045643:DNA Repair Enzymes; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000378:metabolism; D018259:Herpesvirus 1, Human / Q000235:genetics*; D046911:Macromolecular Substances; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D018741:Replication Origin; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C045462:ICP8 protein, Simplexvirus; D046911:Macromolecular Substances; D014764:Viral Proteins; C055789:origin-binding proteins, viral; C064565:UL9 protein, Human herpesvirus 1; D000255:Adenosine Triphosphate; D004247:DNA; C114331:EXO1 protein, human; D005090:Exodeoxyribonucleases; C028450:exodeoxyribonuclease I; D004265:DNA Helicases; D045643:DNA Repair Enzymes