Alteration of Arginine-128 to Alanine Abolishes the Ability of Human 06-Alkylguanine-DNA Alkyltransferase To Repair Methylated DNA but Has No Effect on Its Reaction with 06-Benzylguanine

Sreenivas Kanugula, Karina Goodtzova, Suvarchala Edara, Anthony E. Pegg

Research output: Contribution to journalArticlepeer-review

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Abstract

06-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 06-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 06-benzylguanine. The change of residue tyrosine- 114 to glutamic acid (Y114E) completely abolished the ability to repair 06-methylguanine in DNA in the assays used showing that this was reduced by > 15 000-fold, but the ability to convert 06-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 06-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, Cl45A, 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. The mutant Y114E did not form complexes with either single-stranded or double-stranded Ml3 DNA or with an oligodeoxynucleotide 16-mer in a single-stranded or duplex form. Mutant R128A did form a well retarded complex with double-stranded Ml3 DNA but did not form such a complex with singlestranded Ml3 DNA or with the single-stranded 16-mer. Some complex formation occurred with the double-stranded 16-mer, but this was less stable than the complex formed by control AGT. These results provide direct evidence that the domain of the AGT containing residues 114 and 128 is involved in DNA binding. The results with the mutant R128 further suggest that a single-stranded region is generated during the AGT reaction and that arginine-128 is involved in binding this single-stranded region in a conformation that allows alkyl transfer to occur.

Original languageEnglish (US)
Pages (from-to)7113-7119
Number of pages7
JournalBiochemistry
Volume34
Issue number21
DOIs
StatePublished - May 1995

All Science Journal Classification (ASJC) codes

  • Biochemistry

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