An Initial ATP-Independent Step in the Unwinding of a Herpes Simplex Virus Type I Origin of Replication by a Complex of the Viral Origin-Binding Protein and Single-Strand DNA-Binding Protein

• Published in: Proceedings of the National Academy of Sciences - PNAS 2001-03, Vol.98 (6), p.3024-3028
• Main Authors: He, Xiaodun, Lehman, I. R.
• Format: Article
• Language: English
• Subjects: Adenine; Adenosine triphosphatases; Adenosine Triphosphate - metabolism; AICP8 protein; AUL9 protein; Biochemistry; Biological Sciences; DNA; DNA binding proteins; DNA, Single-Stranded - metabolism; DNA, Viral - metabolism; DNA-Binding Proteins - metabolism; Electrophoresis; Gels; Herpes simplex virus 1; Herpesvirus 1, Human - genetics; Herpesvirus 1, Human - metabolism; Human herpesvirus 1; Humans; ICP8 protein; Molecular dynamics; Oligonucleotides; origin-binding protein; Replication Origin; replication origins; single-stranded DNA-binding protein; Spectrophotometry, Ultraviolet - methods; Thymine; UL9 protein; Viral Proteins - metabolism
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.061028298

Using a spectrophotometric assay that measures the hyperchromicity that accompanies the unwinding of a DNA duplex, we have identified an ATP-independent step in the unwinding of a herpes simplex virus type 1 (HSV-1) origin of replication, Oris, by a complex of the HSV-1 origin binding protein (UL9 protein) and the HSV-1 single-strand DNA binding protein (ICP8). The sequence unwound is the 18-bp A + T-rich segment that links the two high-affinity UL9 protein binding sites, boxes I and II of Oris. P1 nuclease sensitivity of Oris and single-strand DNA-dependent ATPase measurements of the UL9 protein indicate that, at 37°C, the A + T-rich segment is sufficiently single stranded to permit the binding of ICP8. Binding of the UL9 protein to boxes I and II then results in the formation of the UL9 protein-ICP8 complex, that can, in the absence of ATP, promote unwinding of the A + T-rich segment. On addition of ATP, the helicase activity of the UL9 protein-ICP8 complex can unwind boxes I and II, permitting access of the replication machinery to the Oris sequences.