Mutations in Endonuclease V That Affect both Protein-Protein Association and Target Site Location

Courtney Nickell, Robert (Stephen) Lloyd

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

A general mechanism by which proteins locate their target sites within large domains of DNA is a one-dimensional facilitated diffusion process in which the protein scans DNA in a nonspecifically bound state. An electrostatic contribution to this type of mechanism has been previously established. This study was designed to question whether other characteristics of a protein's structure might contribute to the scanning mechanism of target site location. In this regard, T4 endonuclease V was shown to establish an ionic strength dependent monomer-dimer equilibrium in solution. A protein dimer interaction site was postulated to exist along a putative α-helix containing amino acid residues 54-62. The conservative substitutions of Phe-60 → Leu-60 and Phe-59, Phe-60 → Leu-59, Leu-60 resulted in mutant enzymes which remained in the monomeric state independent of the ionic strength of the solution. The target site location mechanism of these mutants has also been altered. Under conditions where wild-type endonuclease V processively scans nontarget DNA, the target location mechanism of the monomeric mutant proteins was shifted toward a less processive search. This decrease in the processivity of the mutants was especially surprising because the nontarget DNA binding affinity was found to be significantly increased. Thus, an additional component of the endonuclease V DNA scanning mechanism appears to be the formation of a stable endonuclease V dimer complex.

Original languageEnglish (US)
Pages (from-to)8638-8648
Number of pages11
JournalBiochemistry
Volume30
Issue number35
DOIs
StatePublished - Sep 1 1991
Externally publishedYes

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Deoxyribonuclease (Pyrimidine Dimer)
Mutation
Dimers
DNA
Proteins
Ionic strength
Osmolar Concentration
Facilitated Diffusion
Scanning
Mutant Proteins
Static Electricity
Electrostatics
Substitution reactions
Monomers
Amino Acids
Enzymes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Mutations in Endonuclease V That Affect both Protein-Protein Association and Target Site Location. / Nickell, Courtney; Lloyd, Robert (Stephen).

In: Biochemistry, Vol. 30, No. 35, 01.09.1991, p. 8638-8648.

Research output: Contribution to journalArticle

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