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 language | English (US) |
|---|---|
| Pages (from-to) | 8796-8803 |
| Number of pages | 8 |
| Journal | Biochemistry |
| Volume | 34 |
| Issue number | 27 |
| DOIs | |
| State | Published - 1995 |
| Externally published | Yes |
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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 journal › Article
}
TY - JOUR
T1 - δ-Elimination by T4 endonuclease V at a thymine dimer site requires a secondary binding event and amino acid Glu-23
AU - Latham, K. A.
AU - Lloyd, Robert (Stephen)
PY - 1995
Y1 - 1995
N2 - 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.
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.
UR - http://www.scopus.com/inward/record.url?scp=0028982164&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0028982164&partnerID=8YFLogxK
U2 - 10.1021/bi00027a031
DO - 10.1021/bi00027a031
M3 - Article
C2 - 7612620
AN - SCOPUS:0028982164
VL - 34
SP - 8796
EP - 8803
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 27
ER -