Reductive methylation of the amino terminus of endonuclease V eradicates catalytic activities. Evidence for an essential role of the amino terminus in the chemical mechanisms of catalysis.

Schrock, Robert D.; Lloyd, R. Stephen

doi:10.1016/s0021-9258(19)47418-8

Cited by 85 publications

(27 citation statements)

References 38 publications

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“…These data indicate that the (/-elimination reaction is a specific enzymatic activity of endonuclease V. d-Elimination by Mutant Enzymes Defective in Thymine Dimer-Specific Nicking. Experiments from this laboratory have demonstrated that the N-terminus of endonuclease V is directly responsible for both the /V-glycosylase and AP lyase activities of the enzyme (Schrock & Lloyd, 1991Dodson et al, 1993). Furthermore, Glu-23, which has been shown by X-ray crystallography to be close to the Nterminus, has also been found to be catalytically important for both activities (Morikawa et al, 1992Doi et al.…”

Section: Resultsmentioning

confidence: 99%

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.delta.-Elimination by T4 Endonuclease V at a Thymine Dimer Site Requires a Secondary Binding Event and Amino Acid Glu-23

Ka¹,

Lloyd²

1995

Biochemistry

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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 beta-elimination mechanism. In addition, high concentrations of endonuclease V have been reported to catalyze the cleavage of the C5'-O-P bond in a reaction referred to as delta-elimination. To better understand the enzymology of endonuclease V, the delta-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 delta-elimination reaction compared to beta-elimination and the ability of unlabeled dimer-containing DNA to compete more efficiently for delta-elimination than beta-elimination indicate that delta-elimination most likely occurs during a separate enzyme encounter with the incised DNA. Previous studies have shown that both the alpha-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 delta-elimination requires Glu-23, but not the primary amine at the N-terminus. In fact, the T2P mutant was much more efficient at promoting delta-elimination than the wild-type enzyme. Besides lending further proof that delta-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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Section: Resultsmentioning

confidence: 99%

“…endonuclease V on C-l' of the 5'-deoxyribose moiety within the dimer (Schrock & Lloyd, 1991;Dodson et al, 1993). These two activities also require the presence of an additional residue, Glu-23 (Hori et al, 1992;Doi et al, 1992;Manuel et al, 1995).…”

mentioning

confidence: 99%

.delta.-Elimination by T4 Endonuclease V at a Thymine Dimer Site Requires a Secondary Binding Event and Amino Acid Glu-23

Ka¹,

Lloyd²

1995

Biochemistry

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“…In T4-endo V, the intermediacy of a Schiff base has been well-documented and the identity of the nucleophilic amino group was determined to be the N-terminus. Reductive methylation of T4-endo V by treatment with formaldehyde and sodium borohydride inactivates the enzyme's glycosylase activity, suggesting the importance of a nucleophilic amino group . Using protease digestion, the site of methylation was determined to be the α-amino group of T4-endo V .…”

Section: B Identification Of Active-site Amino Groups In T4-endo V An...mentioning

confidence: 99%

Chemistry of Glycosylases and Endonucleases Involved in Base-Excision Repair

1998

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ContentsI. Introduction 1221 II. Types of Base-Excision Repair Glycosylases 1223 A. Uracil-DNA Glycosylase (UDG) 1223 B. Mismatch-Specific Thymine-DNA Glycosylase (TDG) and Related Double-Strand Specific Uracil DNA Glycosylase (dsUDG) 1226 C. Alkylated Base Removal 1227 D. Endonuclease III and Related Enzymes 1229 E. Pyrimidine Dimer Glycosylases 1230 F. The FPG Protein (Fapy Glycosylase or MutM) 1231

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“…The most extensively studied enzyme of this type is bacteriophage T4 endonuclease V. This enzyme has a glycosylase activity that hydrolyses the glycosyl bond of the 5‘ pyrimidine in the dimer and an apurinic/apyrimidinic endonuclease activity proceeding through the β-elimination of the 3‘-phosphate of an abasic site . The high resolution X-ray crystal structure of endo V combined with numerous site-directed mutagenesis studies , have allowed the most important residues for binding and catalytic activities to be identified. Recently, an X-ray crystal structure of endonuclease V complexed with duplex DNA containing a thymine dimer was reported …”

Section: Introductionmentioning

confidence: 99%

Computational Simulations of DNA Distortions by a cis,syn-Cyclobutane Thymine Dimer Lesion

et al. 1996

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Results are presented from 500 ps molecular dynamics simulations on the native dodecamer d(CGCGAATTCGCG)2 and the lesioned dodecamer containing a cis,syn-thymine cyclobutane dimer at the TT step. The computations, performed with AMBER4.1, included explicitly represented solvent with periodic boundary conditions applied within the constant temperature and pressure algorithm. Electrostatic interactions were calculated with the particle-mesh Ewald method. Distortions to DNA structure produced by the lesion were found to be localized at the dimer site and include mainly a substantial kink in the helical axis, rolled and tilted base pairs, and weakened hydrogen bonding at the 5‘ base pair of the lesion. A slight change in orientation around the glycosyl bond for the 5‘ thymine of the lesion and highly stiffened deoxyribose rings for both thymine bases were also observed. The global curvature of DNA is increased by about 10° by dimer incorporation. Calculations of H(1‘)-H(6)(pyrimidine) and H(1‘)-H(8)(purine) interproton distances from the performed simulations agree very well with the pattern of NMR NOE signals reported in various dimer containing oligonucleotides, where an interruption of NOE connectivities is found on the 5‘ side of the lesion. Comparison of the pattern of distortions observed at the dimer site with the crystal structure of a complex between dimer-containing DNA and repair enzyme endonuclease V (Cell 1995, 83, 773−782) leads to the hypothesis that dimer recognition may involve a whole pattern of small distortion at the lesion site rather than one particular structural/dynamical feature associated with the lesion.

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Reductive methylation of the amino terminus of endonuclease V eradicates catalytic activities. Evidence for an essential role of the amino terminus in the chemical mechanisms of catalysis.

Cited by 85 publications

References 38 publications

.delta.-Elimination by T4 Endonuclease V at a Thymine Dimer Site Requires a Secondary Binding Event and Amino Acid Glu-23

.delta.-Elimination by T4 Endonuclease V at a Thymine Dimer Site Requires a Secondary Binding Event and Amino Acid Glu-23

Chemistry of Glycosylases and Endonucleases Involved in Base-Excision Repair

Computational Simulations of DNA Distortions by a cis,syn-Cyclobutane Thymine Dimer Lesion

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