Cysteine 111 Affects Coupling of Single-stranded DNA Binding to ATP Hydrolysis in the Herpes Simplex Virus Type-1 Origin-binding Protein

• Published in: The Journal of biological chemistry 2000-01, Vol.275 (4), p.2931-2937
• Main Authors: Sampson, Deborah A., Arana, Mercedes E., Boehmer, Paul E.
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - metabolism; Alanine - chemistry; Alanine - genetics; Amino Acid Sequence; ATP-binding protein; Cysteine - metabolism; Dithiothreitol - pharmacology; DNA helicase; DNA Helicases - chemistry; DNA Helicases - genetics; DNA Helicases - metabolism; DNA Replication; DNA, Single-Stranded - metabolism; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Ethylmaleimide - pharmacology; Herpes simplex virus 1; Hydrolysis; Kinetics; Molecular Sequence Data; Mutagenesis, Site-Directed; origin-binding protein; Protein Binding; Sequence Homology, Amino Acid; single-stranded DNA; UL9 protein; Viral Proteins - chemistry; Viral Proteins - genetics; Viral Proteins - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.275.4.2931

Herpes simplex virus type-1 origin-binding protein (UL9 protein) initiates viral replication by unwinding the origins. It possesses sequence-specific DNA-binding activity, single-stranded DNA-binding activity, DNA helicase activity, and ATPase activity that is strongly stimulated by single-stranded DNA. We have examined the role of cysteines in its action as a DNA helicase. The DNA helicase and DNA-dependent ATPase activities of UL9 protein were stimulated by reducing agent and specifically inactivated by the sulfhydryl-specific reagent N-ethylmaleimide. To identify the cysteine responsible for this phenomenon, a conserved cysteine in the vicinity of the ATP-binding site (cysteine 111) was mutagenized to alanine. UL9C111A protein exhibits defects in its DNA helicase and DNA-dependent ATPase activities and was unable to support origin-specific DNA replication in vivo. A kinetic analysis indicates that these defects are due to the inability of single-stranded DNA to induce high affinity ATP binding in UL9C111A protein. The DNA-dependent ATPase activity of UL9C111A protein is resistant to N-ethylmaleimide, while its DNA helicase activity remains sensitive. Accordingly, sensitivity of UL9 protein to N-ethylmaleimide is due to at least two cysteines. Cysteine 111 is involved in coupling single-stranded DNA binding to ATP-binding and subsequent hydrolysis, while a second cysteine is involved in coupling ATP hydrolysis to DNA unwinding.