Book of Abstracts: Albany 2007
June 19-23 2007
DNA-Binding Mechanism of O6-Alkylguanine-DNA Alkyltransferase: Changes in DNA Twist and Base Stacking
Human O6-alkylguanine-DNA alkyltransferase (AGT) repairs DNA by transfer of alkyl-groups from the O6 positions of guanine residues and O4 positions of thymine residues to residue C145 in its active site. The mechanism by which AGT locates lesion sites embedded in a vast excess of structurally-similar genomic DNA is under investigation in several laboratories. Here, we describe the equilibrium characteristics of DNA·AGT complexes formed with short oligonucleotides and with plasmid DNAs. Sedimentation equilibrium (SE) studies indicate that AGT binds cooperatively to 16-mer oligonucleotides and reaches similar limiting stoichiometries (n = 4 ± 0.5) with both single-stranded (ss) and double stranded (ds) forms. Changes in circular dichroism (CD) at long wavelengths (∼280 nm) and enhanced fluorescence of 2-aminopurine-substituted DNAs suggest that AGT-binding to both forms is accompanied by at least partial unstacking and untwisting of these DNAs, especially in ssDNA. CD analyses suggest that the alpha-helix content of AGT is decreased upon DNA binding. This conformational change is accompanied by a small but reproducible decrease in tryptophan fluorescence intensity but no detectable change in the wavelength of maximum emission. The statistical binding site size for plasmid pUC 19 varies from 60 bp/site for supercoiled (sc) pUC 19 down to 10 bp/site on relaxed circular and linear DNAs. In topoisomerase I relaxation experiments, addition of AGT results in more negatively supercoiled DNA than in control experiments without AGT. These observations are consistent with models in which AGT unwinds the DNA duplex. DNA unwinding may play an important part in the lesion search mechanism or in accessing alkylated DNA bases for repair. Supported by NIH grant GM070662.
Department of Molecular and Cellular Biochemistry