Biological significance of facilitated diffusion in protein-DNA interactions: Applications to T4 endonuclease V-initiated DNA repair

Diane R. Dowd, Robert (Stephen) Lloyd

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59 Citations (Scopus)

Abstract

Facilitated diffusion along nontarget DNA is employed by numerous DNA-inter active proteins to locate specific targets. Until now, the biological significance of DNA scanning has remained elusive. T4 endonuclease V is a DNA repair enzyme which scans nontarget DNA and processively incises DNA at the site of pyrimidine dimers which are produced by exposure to ultraviolet (UV) light. In this study we tested the hypothesis that there exists a direct correlation between the degree of processivity of wild type and mutant endonuclease V molecules and the degree of enhanced UV resistance which is conferred to repair-deficient Eshcerichia coli. This was accomplished by first creating a series of endonuclease V mutants whose in vitro catalytic activities were shown to be very similar to that of the wild type enzyme. However, when the mechanisms by which these enzymes search nontarget DNA for its substrate were analyzed in vitro and in vivo, the mutants displayed varying degrees of nontarget DNA scanning ranging from being nearly as processive as wild type to randomly incising dimers within the DNA population. The ability of these altered endonuclease V molecules to enhance UV survival in DNA repair-deficient E. coli then was assessed. The degree of enhanced UV survival was directly correlated with the level of facilitated diffusion. This is the first conclusive evidence directly relating a reduction of in vivo facilitated diffusion with a change in an observed phenotype. These results support the assertion that the mechanisms which DNA-interactive proteins employ in locating their target sites are of biological significance.

Original languageEnglish (US)
Pages (from-to)3424-3431
Number of pages8
JournalJournal of Biological Chemistry
Volume265
Issue number6
StatePublished - Feb 25 1990
Externally publishedYes

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Facilitated Diffusion
DNA Repair
Repair
DNA
Deoxyribonuclease (Pyrimidine Dimer)
Proteins
DNA Repair-Deficiency Disorders
DNA Repair Enzymes
Pyrimidine Dimers
phage T4 endonuclease V
Scanning
Molecules
Enzymes
Ultraviolet Rays
Dimers
Escherichia coli
Catalyst activity
Phenotype

ASJC Scopus subject areas

  • Biochemistry

Cite this

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abstract = "Facilitated diffusion along nontarget DNA is employed by numerous DNA-inter active proteins to locate specific targets. Until now, the biological significance of DNA scanning has remained elusive. T4 endonuclease V is a DNA repair enzyme which scans nontarget DNA and processively incises DNA at the site of pyrimidine dimers which are produced by exposure to ultraviolet (UV) light. In this study we tested the hypothesis that there exists a direct correlation between the degree of processivity of wild type and mutant endonuclease V molecules and the degree of enhanced UV resistance which is conferred to repair-deficient Eshcerichia coli. This was accomplished by first creating a series of endonuclease V mutants whose in vitro catalytic activities were shown to be very similar to that of the wild type enzyme. However, when the mechanisms by which these enzymes search nontarget DNA for its substrate were analyzed in vitro and in vivo, the mutants displayed varying degrees of nontarget DNA scanning ranging from being nearly as processive as wild type to randomly incising dimers within the DNA population. The ability of these altered endonuclease V molecules to enhance UV survival in DNA repair-deficient E. coli then was assessed. The degree of enhanced UV survival was directly correlated with the level of facilitated diffusion. This is the first conclusive evidence directly relating a reduction of in vivo facilitated diffusion with a change in an observed phenotype. These results support the assertion that the mechanisms which DNA-interactive proteins employ in locating their target sites are of biological significance.",
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