Substitution of basic amino acids within endonuclease V enhances nontarget DNA binding

Courtney Nickell, Wayne F. Anderson, Robert (Stephen) Lloyd

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

Several DNA-interactive proteins, including the DNA repair enzyme T4 endonuclease V, have been shown to locate their target recognition sites utilizing an electrostatically mediated facilitated diffusion mechanism. Previous work indicates that a decrease in the affinity of endonuclease V for nontarget DNA results in an increased nontarget dissociation rate. This study was designed to investigate the effect of an increase in the affinity of endonuclease V for nontarget DNA. Using a working structural model of the enzyme as a guide, the electrostatic character of endonuclease V was altered. Substitution of Thr-7 with Lys-7 resulted in an enzyme with wild type in vitro characteristics. Mutations which increased the positive charge along a proposed solvent-exposed α-helical face had significant effects. The mutants Ala-30, Val-31 → Lys-30, Leu-31 and Asn-37 → Lys-37 displayed wild type in vitro apurinic-specific and dimer-specific nicking activities. Although the processive dimer-specific nicking rate of the Lys-37 mutant resembled that of wild type, the rate of the Lys-30, Leu-31 mutant was reduced by 60%. In addition, the salt concentration range over which these mutants processively nick dimer-containing DNA has been greatly expanded. Both mutants are shown to have an increased affinity for nontarget DNA.

Original languageEnglish (US)
Pages (from-to)5634-5642
Number of pages9
JournalJournal of Biological Chemistry
Volume266
Issue number9
StatePublished - 1991
Externally publishedYes

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Deoxyribonuclease (Pyrimidine Dimer)
Basic Amino Acids
Substitution reactions
Dimers
DNA
Facilitated Diffusion
DNA Repair Enzymes
Structural Models
Enzymes
Static Electricity
Electrostatics
Salts
Mutation
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

Substitution of basic amino acids within endonuclease V enhances nontarget DNA binding. / Nickell, Courtney; Anderson, Wayne F.; Lloyd, Robert (Stephen).

In: Journal of Biological Chemistry, Vol. 266, No. 9, 1991, p. 5634-5642.

Research output: Contribution to journalArticle

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