δ-Elimination by T4 endonuclease V at a thymine dimer site requires a secondary binding event and amino acid Glu-23

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Abstract

Endonuclease V from bacteriophage T4 is a well characterized enzyme that initiates the repair of ultraviolet light induced pyrimidine dimers. Scission of the phosphodiester backbone between the pyrimidines within a dimer, or 3' to an abasic (AP) site, occurs by a β-elimination mechanism. In addition, high concentrations of endonuclease V have been reported to catalyze the cleavage of the C(5')-O-P bond in a reaction referred to as δ-elimination. To better understand the enzymology of endonuclease V, the δ-elimination reaction of the enzyme has been investigated using an oligonucleotide containing a site-specific cis-syn cyclobutane thymine dimer. The slower kinetics of the δ-elimination reaction compared to β-elimination and the ability of unlabeled dimer-containing DNA to compete more efficiently for δ- elimination than β-elimination indicate that δ-elimination most likely occurs during a separate enzyme encounter with the incised DNA. Previous studies have shown that both the α-amino group of the N-terminus and the acidic residue Glu-23 are necessary for the N-glycosylase and AP lyase activities of endonuclease V. Experiments with T2P, E23Q, and E23D mutants, which are defective in pyrimidine dimer-specific nicking, demonstrated that δ-elimination requires Glu-23, but not the primary amine at the N-terminus. In fact, the T2P mutant was much more efficient at promoting δ-elimination than the wild-type enzyme. Besides lending further proof that δ-elimination requires a second encounter between enzyme and DNA, this result may reflect an enhanced binding of the T2P mutant to dimer-containing DNA.

Original languageEnglish (US)
Pages (from-to)8796-8803
Number of pages8
JournalBiochemistry
Volume34
Issue number27
DOIs
StatePublished - 1995
Externally publishedYes

Fingerprint

Pyrimidine Dimers
Deoxyribonuclease (Pyrimidine Dimer)
Amino Acids
Dimers
Enzymes
DNA
DNA-(Apurinic or Apyrimidinic Site) Lyase
Pyrimidines
Ultraviolet Rays
Oligonucleotides
Amines
Repair
phage T4 endonuclease V
Kinetics
Experiments

ASJC Scopus subject areas

  • Biochemistry

Cite this

δ-Elimination by T4 endonuclease V at a thymine dimer site requires a secondary binding event and amino acid Glu-23. / Latham, K. A.; Lloyd, Robert (Stephen).

In: Biochemistry, Vol. 34, No. 27, 1995, p. 8796-8803.

Research output: Contribution to journalArticle

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abstract = "Endonuclease V from bacteriophage T4 is a well characterized enzyme that initiates the repair of ultraviolet light induced pyrimidine dimers. Scission of the phosphodiester backbone between the pyrimidines within a dimer, or 3' to an abasic (AP) site, occurs by a β-elimination mechanism. In addition, high concentrations of endonuclease V have been reported to catalyze the cleavage of the C(5')-O-P bond in a reaction referred to as δ-elimination. To better understand the enzymology of endonuclease V, the δ-elimination reaction of the enzyme has been investigated using an oligonucleotide containing a site-specific cis-syn cyclobutane thymine dimer. The slower kinetics of the δ-elimination reaction compared to β-elimination and the ability of unlabeled dimer-containing DNA to compete more efficiently for δ- elimination than β-elimination indicate that δ-elimination most likely occurs during a separate enzyme encounter with the incised DNA. Previous studies have shown that both the α-amino group of the N-terminus and the acidic residue Glu-23 are necessary for the N-glycosylase and AP lyase activities of endonuclease V. Experiments with T2P, E23Q, and E23D mutants, which are defective in pyrimidine dimer-specific nicking, demonstrated that δ-elimination requires Glu-23, but not the primary amine at the N-terminus. In fact, the T2P mutant was much more efficient at promoting δ-elimination than the wild-type enzyme. Besides lending further proof that δ-elimination requires a second encounter between enzyme and DNA, this result may reflect an enhanced binding of the T2P mutant to dimer-containing DNA.",
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AB - Endonuclease V from bacteriophage T4 is a well characterized enzyme that initiates the repair of ultraviolet light induced pyrimidine dimers. Scission of the phosphodiester backbone between the pyrimidines within a dimer, or 3' to an abasic (AP) site, occurs by a β-elimination mechanism. In addition, high concentrations of endonuclease V have been reported to catalyze the cleavage of the C(5')-O-P bond in a reaction referred to as δ-elimination. To better understand the enzymology of endonuclease V, the δ-elimination reaction of the enzyme has been investigated using an oligonucleotide containing a site-specific cis-syn cyclobutane thymine dimer. The slower kinetics of the δ-elimination reaction compared to β-elimination and the ability of unlabeled dimer-containing DNA to compete more efficiently for δ- elimination than β-elimination indicate that δ-elimination most likely occurs during a separate enzyme encounter with the incised DNA. Previous studies have shown that both the α-amino group of the N-terminus and the acidic residue Glu-23 are necessary for the N-glycosylase and AP lyase activities of endonuclease V. Experiments with T2P, E23Q, and E23D mutants, which are defective in pyrimidine dimer-specific nicking, demonstrated that δ-elimination requires Glu-23, but not the primary amine at the N-terminus. In fact, the T2P mutant was much more efficient at promoting δ-elimination than the wild-type enzyme. Besides lending further proof that δ-elimination requires a second encounter between enzyme and DNA, this result may reflect an enhanced binding of the T2P mutant to dimer-containing DNA.

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