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.
AuthorsRafferty, Joseph A
Wibley, J E
Margison, Geoffrey P
Moody, P C
Douglas, K T
AffiliationCRC Department of Carcinogenesis, Paterson Institute for Cancer Research, Christie Hospital (NHS) Trust, Manchester, UK.
MetadataShow full item record
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.
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. Acta
JournalBiochimica et Biophysica Acta
- Repair of O6-benzylguanine by the Escherichia coli Ada and Ogt and the human O6-alkylguanine-DNA alkyltransferases.
- Authors: Goodtzova K, Kanugula S, Edara S, Pauly GT, Moschel RC, Pegg AE
- Issue date: 1997 Mar 28
- A single amino acid change in human O6-alkylguanine-DNA alkyltransferase decreasing sensitivity to inactivation by O6-benzylguanine.
- Authors: Crone TM, Pegg AE
- Issue date: 1993 Oct 15
- Expression in mammalian cells of the Escherichia coli O6 alkylguanine-DNA-alkyltransferase gene ogt reduces the toxicity of alkylnitrosoureas.
- Authors: Harris LC, Margison GP
- Issue date: 1993 Jun
- Mechanism of inactivation of human O6-alkylguanine-DNA alkyltransferase by O6-benzylguanine.
- Authors: Pegg AE, Boosalis M, Samson L, Moschel RC, Byers TL, Swenn K, Dolan ME
- Issue date: 1993 Nov 16
- Investigation of the role of tyrosine-114 in the activity of human O6-alkylguanine-DNA alkyltranferase.
- Authors: Goodtzova K, Kanugula S, Edara S, Pegg AE
- Issue date: 1998 Sep 8