Uncoupling of nucleotide flipping and DNA bending by the T4 pyrimidine dimer DNA glycosylase

Research output: Contribution to journalArticle

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Abstract

Bacteriophage T4 pyrimidine dimer glycosylase (T4-Pdg) is a base excision repair protein that incises DNA at cyclobutane pyrimidine dimers that are formed as a consequence of exposure to ultraviolet light. Cocrystallization of T4-Pdg with substrate DNA has shown that the adenosine opposite the 5′-thymine of a thymine-thymine (TT) dimer is flipped into an extrahelical conformation and that the DNA backbone is kinked 60° in the enzyme-substrate (ES) complex. To examine the kinetic details of the precatalytic events in the T4-Pdg reaction mechanism, investigations were designed to separately assess nucleotide flipping and DNA bending. The fluorescent adenine base analogue, 2-aminopurine (2-AP), placed opposite an abasic site analogue, tetrahydrofuran, exhibited a 2.8-fold increase in emission intensity when flipped in the ES complex. Using the 2-AP fluorescence signal for nucleotide flipping, kon and koff pre-steady-state kinetic measurements were determined. DNA bending was assessed by fluorescence resonance energy transfer using fluorescent donor-acceptor pairs located at the 5′-ends of oligonucleotides in duplex DNA. The fluorescence intensity of the donor fluorophore was quenched by 15% in the ES complex as a result of an increased efficiency of energy transfer between the labeled ends of the DNA in the bent conformation. Kinetic analyses of the bending signal revealed an off rate that was 2.5-fold faster than the off rate for nucleotide flipping. These results demonstrate that the nucleotide flipping step can be uncoupled from the bending of DNA in the formation of an ES complex.

Original languageEnglish (US)
Pages (from-to)14192-14200
Number of pages9
JournalBiochemistry
Volume45
Issue number47
DOIs
StatePublished - Nov 28 2006

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Pyrimidine Dimers
Nucleotides
DNA
2-Aminopurine
Thymine
Substrates
Enzymes
Kinetics
Conformations
Fluorescence
Nucleic Acid Conformation
Bacteriophage T4
Fluorescence Resonance Energy Transfer
Energy Transfer
Adenine
Bacteriophages
Ultraviolet Rays
deoxyribopyrimidine endonucleosidase
Fluorophores
Oligonucleotides

ASJC Scopus subject areas

  • Biochemistry

Cite this

Uncoupling of nucleotide flipping and DNA bending by the T4 pyrimidine dimer DNA glycosylase. / Walker, Randall K.; McCullough, Amanda; Lloyd, Robert (Stephen).

In: Biochemistry, Vol. 45, No. 47, 28.11.2006, p. 14192-14200.

Research output: Contribution to journalArticle

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