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

Courtney Nickell, Wayne F. Anderson, R. Stephen Lloyd*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

20 Scopus citations


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
Issue number9
StatePublished - 1991

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


Dive into the research topics of 'Substitution of basic amino acids within endonuclease V enhances nontarget DNA binding'. Together they form a unique fingerprint.

Cite this