DNA-binding mechanism of O6-alkylguanine-DNA alkyltransferase: Effects of protein and DNA alkylation on complex stability

Joseph J. Rasimas, Anthony E. Pegg, Michael G. Fried

Research output: Contribution to journalArticle

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Abstract

The mutagenic and cytotoxic effects of many endogenous and exogenous alkylating agents are mitigated by the actions of O6-alkylguanine-DNA alkyltransferase (AGT). In humans this protein protects the integrity of the genome, but it also contributes to the resistance of tumors to DNA-alkylating chemotherapeutic agents. Here we report properties of the interaction between AGT and short DNA oligonucleotides. We show that although AGT sediments as a monomer in the absence of DNA, it binds cooperatively to both single-stranded and double-stranded deoxyribonucleotides. This strong cooperative interaction is only slightly perturbed by active site mutation of AGT or by alkylation of either AGT or DNA. The stoichiometry of complex formation with 16-mer oligonucleotides, assessed by analytical ultracentrifugation and electrophoretic mobility shift assays, is 4:1 on single-stranded and duplex DNA and is unchanged by several active site mutations or by protein or DNA alkylation. These results have significant implications for the mechanisms by which AGT locates and interacts with repairable alkyl lesions to effect DNA repair.

Original languageEnglish (US)
Pages (from-to)7973-7980
Number of pages8
JournalJournal of Biological Chemistry
Volume278
Issue number10
DOIs
StatePublished - Mar 7 2003

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Alkylation
DNA
Alkylating Agents
Proteins
Oligonucleotides
Catalytic Domain
Deoxyribonucleotides
Mutation
Single-Stranded DNA
Ultracentrifugation
Electrophoretic Mobility Shift Assay
DNA Repair
Electrophoretic mobility
DNA alkyltransferase
Genome
Stoichiometry
Tumors
Assays
Sediments
Repair

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

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DNA-binding mechanism of O6-alkylguanine-DNA alkyltransferase : Effects of protein and DNA alkylation on complex stability. / Rasimas, Joseph J.; Pegg, Anthony E.; Fried, Michael G.

In: Journal of Biological Chemistry, Vol. 278, No. 10, 07.03.2003, p. 7973-7980.

Research output: Contribution to journalArticle

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