Site-Directed Mutagenesis of Glutamic Acid 172 to Glutamine Completely Inactivated Human O6-Alkylguanine-DNA-alkyltransferase

J. A. Rafferty; J. Tumelty; M. Skorvaga; R. H. Elder; G. P. Margison; K. T. Douglas

doi:10.1006/bbrc.1994.1226

Site-Directed Mutagenesis of Glutamic Acid 172 to Glutamine Completely Inactivated Human O⁶-Alkylguanine-DNA-alkyltransferase

J. A. Rafferty^*, J. Tumelty, M. Skorvaga, R. H. Elder, G. P. Margison, K. T. Douglas

^*Corresponding author for this work

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Abstract

DNA repair by O⁶-alkylguanine-DNA-alkyltransferase involves the stoichiometric transfer of the O⁶-alkyl group from the guanine lesion to the active-site cysteine residues of the protein. Site-directed mutagenesis of glutamic acid 172 of human O⁶-alkylguanine-DNA-alkyltransferase (EC 2.1.1.63) to glutamine totally abolished the alkyltransferase activity of the protein. This suggests that glutamic acid 172 is crucial to the alkyl transfer. It may act as a general acid (as CO₂H) or base (as CO₂^-), or have a role as a component of a salt-link (-CO₂^-.....⁺N-), vital for the structural integrity of the active site. This is the first mutational inactivation of a protein in this family of DNA repair molecules by means of a residue change outside the highly conserved pentet (PCHRV) which includes the active-site cysteine.

Original language	English
Pages (from-to)	285-291
Number of pages	7
Journal	Biochemical and Biophysical Research Communications
Volume	199
Issue number	1
DOIs	https://doi.org/10.1006/bbrc.1994.1226
Publication status	Published - 28 Feb 1994

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title = "Site-Directed Mutagenesis of Glutamic Acid 172 to Glutamine Completely Inactivated Human O6-Alkylguanine-DNA-alkyltransferase",

abstract = "DNA repair by O6-alkylguanine-DNA-alkyltransferase involves the stoichiometric transfer of the O6-alkyl group from the guanine lesion to the active-site cysteine residues of the protein. Site-directed mutagenesis of glutamic acid 172 of human O6-alkylguanine-DNA-alkyltransferase (EC 2.1.1.63) to glutamine totally abolished the alkyltransferase activity of the protein. This suggests that glutamic acid 172 is crucial to the alkyl transfer. It may act as a general acid (as CO2H) or base (as CO2-), or have a role as a component of a salt-link (-CO2-.....+N-), vital for the structural integrity of the active site. This is the first mutational inactivation of a protein in this family of DNA repair molecules by means of a residue change outside the highly conserved pentet (PCHRV) which includes the active-site cysteine.",

author = "Rafferty, {J. A.} and J. Tumelty and M. Skorvaga and Elder, {R. H.} and Margison, {G. P.} and Douglas, {K. T.}",

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T1 - Site-Directed Mutagenesis of Glutamic Acid 172 to Glutamine Completely Inactivated Human O6-Alkylguanine-DNA-alkyltransferase

AU - Rafferty, J. A.

AU - Tumelty, J.

AU - Skorvaga, M.

AU - Elder, R. H.

AU - Margison, G. P.

AU - Douglas, K. T.

PY - 1994/2/28

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N2 - DNA repair by O6-alkylguanine-DNA-alkyltransferase involves the stoichiometric transfer of the O6-alkyl group from the guanine lesion to the active-site cysteine residues of the protein. Site-directed mutagenesis of glutamic acid 172 of human O6-alkylguanine-DNA-alkyltransferase (EC 2.1.1.63) to glutamine totally abolished the alkyltransferase activity of the protein. This suggests that glutamic acid 172 is crucial to the alkyl transfer. It may act as a general acid (as CO2H) or base (as CO2-), or have a role as a component of a salt-link (-CO2-.....+N-), vital for the structural integrity of the active site. This is the first mutational inactivation of a protein in this family of DNA repair molecules by means of a residue change outside the highly conserved pentet (PCHRV) which includes the active-site cysteine.

AB - DNA repair by O6-alkylguanine-DNA-alkyltransferase involves the stoichiometric transfer of the O6-alkyl group from the guanine lesion to the active-site cysteine residues of the protein. Site-directed mutagenesis of glutamic acid 172 of human O6-alkylguanine-DNA-alkyltransferase (EC 2.1.1.63) to glutamine totally abolished the alkyltransferase activity of the protein. This suggests that glutamic acid 172 is crucial to the alkyl transfer. It may act as a general acid (as CO2H) or base (as CO2-), or have a role as a component of a salt-link (-CO2-.....+N-), vital for the structural integrity of the active site. This is the first mutational inactivation of a protein in this family of DNA repair molecules by means of a residue change outside the highly conserved pentet (PCHRV) which includes the active-site cysteine.

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JO - Biochemical and Biophysical Research Communications

JF - Biochemical and Biophysical Research Communications

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Site-Directed Mutagenesis of Glutamic Acid 172 to Glutamine Completely Inactivated Human O⁶-Alkylguanine-DNA-alkyltransferase

Abstract

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