Cysteine 111 affects coupling of single-stranded DNA binding to ATP hydrolysis in the herpes simplex virus type-1 origin-binding protein

Deborah A. Sampson, Mercedes E. Arana, Paul E Boehmer

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)2931-2937
Number of pages7
JournalJournal of Biological Chemistry
Volume275
Issue number4
DOIs
StatePublished - Jan 28 2000
Externally publishedYes

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Single-Stranded DNA
Human Herpesvirus 1
Viruses
DNA Helicases
Cysteine
Hydrolysis
Carrier Proteins
Adenosine Triphosphate
Ethylmaleimide
Adenosine Triphosphatases
Proteins
DNA
Sulfhydryl Reagents
Defects
Replication Origin
Reducing Agents
Viral Proteins
DNA Replication
Alanine
Binding Sites

ASJC Scopus subject areas

  • Biochemistry

Cite this

Cysteine 111 affects coupling of single-stranded DNA binding to ATP hydrolysis in the herpes simplex virus type-1 origin-binding protein. / Sampson, Deborah A.; Arana, Mercedes E.; Boehmer, Paul E.

In: Journal of Biological Chemistry, Vol. 275, No. 4, 28.01.2000, p. 2931-2937.

Research output: Contribution to journalArticle

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