T4 endonuclease V exists in solution as a monomer and binds to target sites as a monomer

Katherine Atkins Latham, Surendran Rajendran, J. Russ Carmical, James Lee, R. Stephen Lloyd

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Endonuclease V, a N-glycosylase/lyase from T4 bacteriophage that initiates the repair of cyclobutane pyrimidine dimers in DNA, has been reported to form a monomer-dimer equilibrium in solution [Nickell and Lloyd (1991) Biochemistry 30, 8638], although the enzyme has only been crystallized in the absence of substrate as a monomer [Morikawa et al. (1992) Science 256, 523]. In this study, analytical gel filtration and sedimentation equilibrium techniques were used to rigorously characterize the association state of the enzyme in solution. In contrast to the previous report, at 100 mM KCl endonuclease V was found to exist predominantly as a monomer in solution by both of these techniques; no evidence for dimerization was seen. To characterize the oligomeric state of the enzyme at its target sites on DNA, the enzyme was bound to oligonucleotides containing a single site-specific pyrimidine dimer or tetrahydrofuran residue. These complexes were analyzed by nondenaturing gel electrophoresis at various acrylamide concentrations in order to determine the molecular weights of the enzyme-DNA complexes. The results from these experiments demonstrate that endonuclease V binds to cyclobutane pyrimidine dimer and tetrahydrofuran site containing DNA as a monomer.

Original languageEnglish (US)
Pages (from-to)324-334
Number of pages11
JournalBiochimica et Biophysica Acta - Protein Structure and Molecular Enzymology
Volume1292
Issue number2
DOIs
StatePublished - Feb 8 1996

Fingerprint

Deoxyribonuclease (Pyrimidine Dimer)
Pyrimidine Dimers
Monomers
Enzymes
DNA
Gels
Bacteriophage T4
Biochemistry
Bacteriophages
Lyases
Dimerization
Acrylamide
Electrophoresis
Sedimentation
Oligonucleotides
Dimers
Gel Chromatography
Repair
Molecular Weight
Molecular weight

Keywords

  • Cyclobutane pyrimidine dimer
  • Endonuclease V
  • N-glycosylase
  • Protein-DNA interaction
  • Tetrahydrofuran

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Structural Biology
  • Biophysics

Cite this

T4 endonuclease V exists in solution as a monomer and binds to target sites as a monomer. / Latham, Katherine Atkins; Rajendran, Surendran; Carmical, J. Russ; Lee, James; Lloyd, R. Stephen.

In: Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology, Vol. 1292, No. 2, 08.02.1996, p. 324-334.

Research output: Contribution to journalArticle

Latham, Katherine Atkins ; Rajendran, Surendran ; Carmical, J. Russ ; Lee, James ; Lloyd, R. Stephen. / T4 endonuclease V exists in solution as a monomer and binds to target sites as a monomer. In: Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology. 1996 ; Vol. 1292, No. 2. pp. 324-334.
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