ALTERATION OF ARGININE-128 TO ALANINE ABOLISHES THE ABILITY OF HUMAN O-6-ALKYLGUANINE-DNA ALKYLTRANSFERASE TO REPAIR METHYLATED DNA BUT HASNO EFFECT ON ITS REACTION WITH O-6-BENZYLGUANINE

O-6-Alkylguanine-DNA alkyltransferase (AGT) is a DNA repair protein th at removes the promutagenic O-6-methylguanine lesion from DNA. In orde r to obtain more information about the mechanism of action of AGT, two conserved residues in a putative DNA binding domain were changed by s ite-directed mutagenesis, and the abilities of the mutant proteins to bind to DNA, to repair methylated DNA, and to convert O-6-benzylguanin e to guanine were examined. The alteration of arginine-128 to alanine (R128A) reduced the AGT activity toward methylated DNA substrates by a factor of more than 1000 but did not decrease the rate of reaction wi th O-6-benzylguanine. The change of residue tyrosine-114 to glutamic a cid (Y114E) completely abolished the ability to repair O-6-methylguani ne in DNA in the assays used showing that this was reduced by >15 000- fold, but the ability to convert O-6-benzylguanine to guanine was redu ced by only 60-fold. Alteration of this residue to alanine (Y114A) red uced activity toward methylated DNA by >1000-fold and toward O-6-benzy lguanine by about 80-fold. Neither the R128A nor the Y114E mutant AGT were able to compete with the control AGT for the repair of methylated DNA whereas the inactive mutant, C145A, in which the cysteine accepto r site is changed to alanine, competed effectively in this assay. Thes e results suggest that the residues arginine-128 and tyrosine-114 are involved in the DNA binding properties of the AGT. The ability of the AGT proteins to form stable complexes with DNA was therefore examined by measuring the retardation of DNA during electrophoresis. The mutant Y114E did not form complexes with either single-stranded or double-st randed M13 DNA or with an oligodeoxynucleotide 16-mer in a single-stra nded or duplex form. Mutant R128A did form a well retarded complex wit h double-stranded M13 DNA but did not form such a complex with single- stranded M13 DNA or with the single-stranded 16-mer. Some complex form ation occurred with the double-stranded 16-mer, but this was less stab le than the complex formed by control AGT. These results provide direc t evidence that the domain of the AGT containing residues 114 and 128 is involved in DNA binding. The results with the mutant R128 further s uggest that a single-stranded region is generated during the AGT react ion and that arginine-128 is involved in binding this single-stranded region in a conformation that allows alkyl transfer to occur.