THE POTENTIAL ROLE OF GLYCINE-160 OF HUMAN O-6-ALKYLGUANINE-DNA ALKYLTRANSFERASE IN REACTION WITH O-6-BENZYLGUANINE AS DETERMINED BY SITE-DIRECTED MUTAGENESIS AND MOLECULAR MODELING COMPARISONS

O-6-Alkylguanine DNA-alkyltransferase (ATase) repairs toxic, mutagenic and carcinogenic O-6-alkylguanine (O-6-alkG) lesions in DNA by a high ly conserved reaction involving the stoichiometric transfer of the alk yl group to the active centre cysteine residue of the ATase protein. I n the Escherichia coli Ada ATase, which is effectively refactory to in hibition by O-6-benzylguanine (O-6-BzG), the residue corresponding to glycine-160 (G160) for the mammalian proteins of this class is replace d by a tryptophan (W). Therefore, to investigate the potential role of the G160 of the human ATase (hAT) protein in determining sensitivity to O-6-BzG, site-directed mutagenesis was used to produce a mutant pro tein (hATG160W) substituted at position 160 with a W residue. The hATG 160W mutant was found to be stably expressed and was 3- and 5-fold mor e sensitive than hAT to inactivation by O-6-BzG, in the absence and pr esence of additional calf-thymus DNA respectively. A similar, DNA depe ndent increased sensitivity of the hATG160W mutant relative to wild-ty pe was also found for O-6-methylguanine mediated inactivation. The pot ential role of the W160 residue in stabilising the binding of the O-6- alkG to the protein is discussed in terms of a homology model of the s tructure of hAT. The region occupied by G/W-160 forms the site of a pu tative hinge that could be important in the conformational change that is likely to occur on DNA binding. Three sequence motifs have been id entified in this region which may influence O-6-BzG access to the acti ve site; YSGG or YSGGG in mammals (YAGG in E. coli Ogt, YAGS in Dat fr om Bacillus subtilis), YRWG in E. coli Ada and Salmonella typhimurium (but YKWS in Saccharomyces cerevisiae) or YRGGF in AdaB from B. Subtil is. Finally, conformational and stereoelectronic analysis of the putat ive transition states for the alkyl transfer from a series of inactiva tors of hAT, including O-6-BzG was undertaken to rationalise the unexp ected weak inhibition shown by the alpha-pi-unsaturated electrophiles. (C) 1997 Elsevier Science B.V.