title: Structural and biophysical characterization of the proteins interacting with the herpes simplex virus 1 origin of replication.
abstract: The C terminus of the herpes simplex virus type 1 origin-binding protein, UL9ct, interacts directly with the viral single-stranded DNA-binding protein ICP8. We show that a 60-amino acid C-terminal deletion mutant of ICP8 (ICP8DeltaC) also binds very strongly to UL9ct. Using small angle x-ray scattering, the low resolution solution structures of UL9ct alone, in complex with ICP8DeltaC, and in complex with a 15-mer double-stranded DNA containing Box I of the origin of replication are described. Size exclusion chromatography, analytical ultracentrifugation, and electrophoretic mobility shift assays, backed up by isothermal titration calorimetry measurements, are used to show that the stoichiometry of the UL9ct-dsDNA15-mer complex is 2:1 at micromolar protein concentrations. The reaction occurs in two steps with initial binding of UL9ct to DNA (Kd approximately 6 nM) followed by a second binding event (Kd approximately 0.8 nM). It is also shown that the stoichiometry of the ternary UL9ct-ICP8DeltaC-dsDNA15-mer complex is 2:1:1, at the concentrations used in the different assays. Electron microscopy indicates that the complex assembled on the extended origin, oriS, rather than Box I alone, is much larger. The results are consistent with a simple model whereby a conformational switch of the UL9 DNA-binding domain upon binding to Box I allows the recruitment of a UL9-ICP8 complex by interaction between the UL9 DNA-binding domains.
authors.affiliation: From European Molecular Biology Laboratory, Hamburg Outstation, D-22603 Hamburg, Germany; Biophysics Laboratories, Institute of Biomedical and Biomolecular Sciences, University of Portsmouth, Portsmouth PO1 2DT, United Kingdom.
authors.forename: Ioannis
authors.initials: I
authors.lastname: Manolaridis
authors.affiliation: From European Molecular Biology Laboratory, Hamburg Outstation, D-22603 Hamburg, Germany.
authors.forename: Eleni
authors.initials: E
authors.lastname: Mumtsidu
authors.affiliation: From European Molecular Biology Laboratory, Hamburg Outstation, D-22603 Hamburg, Germany; Institute of Crystallography, Russian Academy of Sciences, Leninsky pr. 59, 117333 Moscow, Russia.
authors.forename: Peter
authors.initials: P
authors.lastname: Konarev
authors.affiliation: Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599-7295.
authors.forename: Alexander M
authors.initials: AM
authors.lastname: Makhov
authors.affiliation: From European Molecular Biology Laboratory, Hamburg Outstation, D-22603 Hamburg, Germany.
authors.forename: Stephen W
authors.initials: SW
authors.lastname: Fullerton
authors.affiliation: Institute of Pharmacy, Martin Luther University Halle-Wittenberg, D-06120 Halle, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Sinz
authors.affiliation: Institute of Pharmacy, Martin Luther University Halle-Wittenberg, D-06120 Halle, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Kalkhof
authors.affiliation: Biophysics Laboratories, Institute of Biomedical and Biomolecular Sciences, University of Portsmouth, Portsmouth PO1 2DT, United Kingdom.
authors.forename: John E
authors.initials: JE
authors.lastname: McGeehan
authors.affiliation: Biophysics Laboratories, Institute of Biomedical and Biomolecular Sciences, University of Portsmouth, Portsmouth PO1 2DT, United Kingdom.
authors.forename: Peter D
authors.initials: PD
authors.lastname: Cary
authors.affiliation: Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599-7295.
authors.forename: Jack D
authors.initials: JD
authors.lastname: Griffith
authors.affiliation: From European Molecular Biology Laboratory, Hamburg Outstation, D-22603 Hamburg, Germany; Institute of Crystallography, Russian Academy of Sciences, Leninsky pr. 59, 117333 Moscow, Russia.
authors.forename: Dmitri
authors.initials: D
authors.lastname: Svergun
authors.affiliation: Biophysics Laboratories, Institute of Biomedical and Biomolecular Sciences, University of Portsmouth, Portsmouth PO1 2DT, United Kingdom.
authors.forename: Geoff G
authors.initials: GG
authors.lastname: Kneale
authors.affiliation: From European Molecular Biology Laboratory, Hamburg Outstation, D-22603 Hamburg, Germany. Electronic address: tucker@embl-hamburg.de.
authors.forename: Paul A
authors.initials: PA
authors.lastname: Tucker