Alteration of arginine-128 to alanine abolishes the ability of human O6-alkylguanine-DNA alkyltransferase to repair methylated DNA but has no effect on its reaction with O6-benzylguanine.
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Kanugula S, Goodtzova K, Edara S, Pegg AE
Alteration of arginine-128 to alanine abolishes the ability of human O6-alkylguanine-DNA alkyltransferase to repair methylated DNA but has no effect on its reaction with O6-benzylguanine.
Biochemistry. 1995 May 30;34(21):7113-9.
- PubMed ID
- 7766621 [ View in PubMed]
- Abstract
O6-Alkylguanine-DNA alkyltransferase (AGT) is a DNA repair protein that removes the promutagenic O6-methylguanine lesion from DNA. In order to obtain more information about the mechanism of action of AGT, two conserved residues in a putative DNA binding domain were changed by site-directed mutagenesis, and the abilities of the mutant proteins to bind to DNA, to repair methylated DNA, and to convert O6-benzylguanine 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 with O6-benzylguanine. The change of residue tyrosine-114 to glutamic acid (Y114E) completely abolished the ability to repair O6-methylguanine in DNA in the assays used showing that this was reduced by > 15,000-fold, but the ability to convert O6-benzylguanine to guanine was reduced by only 60-fold. Alteration of this residue to alanine (Y114A) reduced activity toward methylated DNA by > 1000-fold and toward O6-benzylguanine 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 acceptor site is changed to alanine, competed effectively in this assay. These 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.(ABSTRACT TRUNCATED AT 250 WORDS)