Escherichia coli apurinic-apyrimidinic endonucleases enhance the turnover of the adenine glycosylase MutY with G

A substrates

Mary Ann Pope, Silvia L. Porello, Sheila S. David

Research output: Contribution to journalArticle

47 Citations (Scopus)

Abstract

The DNA repair enzyme MutY plays an important role in the prevention of DNA mutations resulting from the presence of the oxidatively damaged lesion 7,8-dihydro8-oxo-2′-deoxyguanosine (OG). MutY is a base excision repair (BER) glycosylase that removes misincorporated adenine residues from OG:A mispairs, as well as G:A and C:A mispairs. We have previously shown that, under conditions of low MutY concentrations relative to an OG:A or G:A substrate, the time course of the adenine glycosylase reaction exhibits biphasic kinetic behavior due to slow release of the DNA product by MutY. The dissociation of MutY from its product may require the recruitment of other proteins from the BER pathway, such as an apurinic-apyrimidinic (AP) endonuclease, as turnover-enhancing cofactors. The effect of the AP endonucleases endonuclease IV (Endo IV), exonuclease III (Exo III), and Ape1 on the reaction kinetics of MutY with G:A- and OG:A-containing substrates was investigated. The effect of the glycosylases UDG and MutM and the DNA polymerase pol I was also characterized. Endo IV and Exo III, unlike Ape1, UDG, and pol I, greatly enhance the rate of product release with a G:A substrate, whereas the rate constant for the adenine removal step remains unchanged. Furthermore, the turnover rate with a truncated form of MutY, Stop 225, which lacks 125 amino acids of the C terminus, is unaffected by the presence of Endo IV or Exo III. These results constitute the first evidence of an interaction between the MutY-product DNA complex and Endo IV or Exo III. Furthermore, they suggest a role for the C-terminal domain of MutY in mediating this interaction.

Original languageEnglish (US)
Pages (from-to)22605-22615
Number of pages11
JournalJournal of Biological Chemistry
Volume277
Issue number25
DOIs
StatePublished - Jun 21 2002
Externally publishedYes

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Deoxyribonuclease IV (Phage T4-Induced)
DNA-(Apurinic or Apyrimidinic Site) Lyase
Endonucleases
Escherichia coli
DNA Polymerase I
Adenine
Substrates
DNA Repair
DNA
Repair
DNA Repair Enzymes
Deoxyribonuclease I
DNA-Directed DNA Polymerase
Reaction kinetics
Rate constants
Amino Acids
Mutation
Kinetics
G-substrate
mutY adenine glycosylase

ASJC Scopus subject areas

  • Biochemistry

Cite this

Escherichia coli apurinic-apyrimidinic endonucleases enhance the turnover of the adenine glycosylase MutY with G : A substrates. / Pope, Mary Ann; Porello, Silvia L.; David, Sheila S.

In: Journal of Biological Chemistry, Vol. 277, No. 25, 21.06.2002, p. 22605-22615.

Research output: Contribution to journalArticle

Pope, Mary Ann ; Porello, Silvia L. ; David, Sheila S. / Escherichia coli apurinic-apyrimidinic endonucleases enhance the turnover of the adenine glycosylase MutY with G : A substrates. In: Journal of Biological Chemistry. 2002 ; Vol. 277, No. 25. pp. 22605-22615.
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abstract = "The DNA repair enzyme MutY plays an important role in the prevention of DNA mutations resulting from the presence of the oxidatively damaged lesion 7,8-dihydro8-oxo-2′-deoxyguanosine (OG). MutY is a base excision repair (BER) glycosylase that removes misincorporated adenine residues from OG:A mispairs, as well as G:A and C:A mispairs. We have previously shown that, under conditions of low MutY concentrations relative to an OG:A or G:A substrate, the time course of the adenine glycosylase reaction exhibits biphasic kinetic behavior due to slow release of the DNA product by MutY. The dissociation of MutY from its product may require the recruitment of other proteins from the BER pathway, such as an apurinic-apyrimidinic (AP) endonuclease, as turnover-enhancing cofactors. The effect of the AP endonucleases endonuclease IV (Endo IV), exonuclease III (Exo III), and Ape1 on the reaction kinetics of MutY with G:A- and OG:A-containing substrates was investigated. The effect of the glycosylases UDG and MutM and the DNA polymerase pol I was also characterized. Endo IV and Exo III, unlike Ape1, UDG, and pol I, greatly enhance the rate of product release with a G:A substrate, whereas the rate constant for the adenine removal step remains unchanged. Furthermore, the turnover rate with a truncated form of MutY, Stop 225, which lacks 125 amino acids of the C terminus, is unaffected by the presence of Endo IV or Exo III. These results constitute the first evidence of an interaction between the MutY-product DNA complex and Endo IV or Exo III. Furthermore, they suggest a role for the C-terminal domain of MutY in mediating this interaction.",
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AB - The DNA repair enzyme MutY plays an important role in the prevention of DNA mutations resulting from the presence of the oxidatively damaged lesion 7,8-dihydro8-oxo-2′-deoxyguanosine (OG). MutY is a base excision repair (BER) glycosylase that removes misincorporated adenine residues from OG:A mispairs, as well as G:A and C:A mispairs. We have previously shown that, under conditions of low MutY concentrations relative to an OG:A or G:A substrate, the time course of the adenine glycosylase reaction exhibits biphasic kinetic behavior due to slow release of the DNA product by MutY. The dissociation of MutY from its product may require the recruitment of other proteins from the BER pathway, such as an apurinic-apyrimidinic (AP) endonuclease, as turnover-enhancing cofactors. The effect of the AP endonucleases endonuclease IV (Endo IV), exonuclease III (Exo III), and Ape1 on the reaction kinetics of MutY with G:A- and OG:A-containing substrates was investigated. The effect of the glycosylases UDG and MutM and the DNA polymerase pol I was also characterized. Endo IV and Exo III, unlike Ape1, UDG, and pol I, greatly enhance the rate of product release with a G:A substrate, whereas the rate constant for the adenine removal step remains unchanged. Furthermore, the turnover rate with a truncated form of MutY, Stop 225, which lacks 125 amino acids of the C terminus, is unaffected by the presence of Endo IV or Exo III. These results constitute the first evidence of an interaction between the MutY-product DNA complex and Endo IV or Exo III. Furthermore, they suggest a role for the C-terminal domain of MutY in mediating this interaction.

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