The potential role of glycine-160 of human O6-alkylguanine-DNA alkyltransferase in reaction with O6-benzylguanine as determined by site-directed mutagenesis and molecular modelling comparisons.

2.50
Hdl Handle:
http://hdl.handle.net/10541/94928
Title:
The potential role of glycine-160 of human O6-alkylguanine-DNA alkyltransferase in reaction with O6-benzylguanine as determined by site-directed mutagenesis and molecular modelling comparisons.
Authors:
Rafferty, Joseph A; Wibley, J E; Speers, P; Hickson, Ian; Margison, Geoffrey P; Moody, P C; Douglas, K T
Abstract:
O6-Alkylguanine DNA-alkyltransferase (ATase) repairs toxic, mutagenic and carcinogenic O6-alkylguanine (O6-alkG) lesions in DNA by a highly conserved reaction involving the stoichiometric transfer of the alkyl group to the active centre cysteine residue of the ATase protein. In the Escherichia coli Ada ATase, which is effectively refactory to inhibition by O6-benzylguanine (O6-BzG), the residue corresponding to glycine-160 (G160) for the mammalian proteins of this class is replaced by a tryptophan (W). Therefore, to investigate the potential role of the G160 of the human ATase (hAT) protein in determining sensitivity to O6-BzG, site-directed mutagenesis was used to produce a mutant protein (hATG160W) substituted at position 160 with a W residue. The hATG160W mutant was found to be stably expressed and was 3- and 5-fold more sensitive than hAT to inactivation by O6-BzG, in the absence and presence of additional calf-thymus DNA respectively. A similar, DNA dependent increased sensitivity of the hATG160W mutant relative to wild-type was also found for O6-methylguanine mediated inactivation. The potential role of the W160 residue in stabilising the binding of the O6-alkG to the protein is discussed in terms of a homology model of the structure of hAT. The region occupied by G/W-160 forms the site of a putative hinge that could be important in the conformational change that is likely to occur on DNA binding. Three sequence motifs have been identified in this region which may influence O6-BzG access to the active site; YSGG or YSGGG in mammals (YAGG in E. coli Ogt, YAGS in Dat from Bacillus subtilis), YRWG in E. coli Ada and Salmonella typhimurium (but YKWS in Saccharomyces cerevisiae) or YRGGF in AdaB from B. Subtilis. Finally,conformational and stereoelectronic analysis of the putative transition states for the alkyl transfer from a series of inactivators of hAT, including O6-BzG was undertaken to rationalise the unexpected weak inhibition shown by the alpha-pi-unsaturated electrophiles.
Affiliation:
CRC Department of Carcinogenesis, Paterson Institute for Cancer Research, Christie Hospital (NHS) Trust, Manchester, UK.
Citation:
The potential role of glycine-160 of human O6-alkylguanine-DNA alkyltransferase in reaction with O6-benzylguanine as determined by site-directed mutagenesis and molecular modelling comparisons. 1997, 1342 (1):90-102 Biochim. Biophys. Acta
Journal:
Biochimica et Biophysica Acta
Issue Date:
26-Sep-1997
URI:
http://hdl.handle.net/10541/94928
PubMed ID:
9366274
Type:
Article
Language:
en
ISSN:
0006-3002
Appears in Collections:
All Paterson Institute for Cancer Research

Full metadata record

DC FieldValue Language
dc.contributor.authorRafferty, Joseph Aen
dc.contributor.authorWibley, J Een
dc.contributor.authorSpeers, Pen
dc.contributor.authorHickson, Ianen
dc.contributor.authorMargison, Geoffrey Pen
dc.contributor.authorMoody, P Cen
dc.contributor.authorDouglas, K Ten
dc.date.accessioned2010-03-24T16:05:22Z-
dc.date.available2010-03-24T16:05:22Z-
dc.date.issued1997-09-26-
dc.identifier.citationThe potential role of glycine-160 of human O6-alkylguanine-DNA alkyltransferase in reaction with O6-benzylguanine as determined by site-directed mutagenesis and molecular modelling comparisons. 1997, 1342 (1):90-102 Biochim. Biophys. Actaen
dc.identifier.issn0006-3002-
dc.identifier.pmid9366274-
dc.identifier.urihttp://hdl.handle.net/10541/94928-
dc.description.abstractO6-Alkylguanine DNA-alkyltransferase (ATase) repairs toxic, mutagenic and carcinogenic O6-alkylguanine (O6-alkG) lesions in DNA by a highly conserved reaction involving the stoichiometric transfer of the alkyl group to the active centre cysteine residue of the ATase protein. In the Escherichia coli Ada ATase, which is effectively refactory to inhibition by O6-benzylguanine (O6-BzG), the residue corresponding to glycine-160 (G160) for the mammalian proteins of this class is replaced by a tryptophan (W). Therefore, to investigate the potential role of the G160 of the human ATase (hAT) protein in determining sensitivity to O6-BzG, site-directed mutagenesis was used to produce a mutant protein (hATG160W) substituted at position 160 with a W residue. The hATG160W mutant was found to be stably expressed and was 3- and 5-fold more sensitive than hAT to inactivation by O6-BzG, in the absence and presence of additional calf-thymus DNA respectively. A similar, DNA dependent increased sensitivity of the hATG160W mutant relative to wild-type was also found for O6-methylguanine mediated inactivation. The potential role of the W160 residue in stabilising the binding of the O6-alkG to the protein is discussed in terms of a homology model of the structure of hAT. The region occupied by G/W-160 forms the site of a putative hinge that could be important in the conformational change that is likely to occur on DNA binding. Three sequence motifs have been identified in this region which may influence O6-BzG access to the active site; YSGG or YSGGG in mammals (YAGG in E. coli Ogt, YAGS in Dat from Bacillus subtilis), YRWG in E. coli Ada and Salmonella typhimurium (but YKWS in Saccharomyces cerevisiae) or YRGGF in AdaB from B. Subtilis. Finally,conformational and stereoelectronic analysis of the putative transition states for the alkyl transfer from a series of inactivators of hAT, including O6-BzG was undertaken to rationalise the unexpected weak inhibition shown by the alpha-pi-unsaturated electrophiles.en
dc.language.isoenen
dc.subject.meshAmino Acid Substitution-
dc.subject.meshBacterial Proteins-
dc.subject.meshBinding Sites-
dc.subject.meshCalorimetry-
dc.subject.meshEscherichia coli-
dc.subject.meshEscherichia coli Proteins-
dc.subject.meshGlycine-
dc.subject.meshGuanine-
dc.subject.meshHumans-
dc.subject.meshKinetics-
dc.subject.meshModels, Molecular-
dc.subject.meshMutagenesis, Site-Directed-
dc.subject.meshO(6)-Methylguanine-DNA Methyltransferase-
dc.subject.meshProtein Conformation-
dc.subject.meshRecombinant Proteins-
dc.subject.meshThermodynamics-
dc.subject.meshTranscription Factors-
dc.titleThe potential role of glycine-160 of human O6-alkylguanine-DNA alkyltransferase in reaction with O6-benzylguanine as determined by site-directed mutagenesis and molecular modelling comparisons.en
dc.typeArticleen
dc.contributor.departmentCRC Department of Carcinogenesis, Paterson Institute for Cancer Research, Christie Hospital (NHS) Trust, Manchester, UK.en
dc.identifier.journalBiochimica et Biophysica Actaen
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