Book of Abstracts: Albany 2005
Surprising DNA Binding Activities of O6-Alkylguanine-DNA Alkyltransferase (AGT)
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 while AGT sediments as a monomer in the absence of DNA, it binds with high cooperativity to both single-stranded and double-stranded deoxyribonucleotides. This result is surprising in view of the 1:1 binding mechanism found in the crystalline AGT-DNA complex. The strongly-cooperative interaction is only slightly perturbed by changes in buffer composition, active site mutation of AGT or by alkylation of either AGT or DNA. These results have significant implications for the mechanisms by which AGT locates and interacts with O6-alkylguanine lesions to effect DNA repair. Supported by NIH grant GM070622.
Binding of AGT to 16 nt single-stranded and 16bp duplex DNAs. Reactions were carried out at 21°C in 10 mM Tris (pH 7.4), 100 mM KCl, 1 mM DTT. The smooth curves are fits to Record?s modification of the McGhee-von Hippel equation, which accounts for statistical end-effects (1). The similarities in stoichiometry (n), association constant (K) and cooperativity (ω) suggest that the binding mechanism is nearly independent of DNA structure.
References and Footnotes
Michael G. Fried*
Department of Molecular and Cellular Biochemistry