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.