Repair of O6-methylguanine adducts in human telomeric G-quadruplex DNA by O6-alkylguanine-DNA alkyltransferase.
Bottom Line: Its functions with short single-stranded and duplex substrates have been characterized, but its ability to act on other DNA structures remains poorly understood.Here, we examine the functions of this enzyme on O(6)-methylguanine (6mG) adducts in the four-stranded structure of the human telomeric G-quadruplex.This distinction may reflect differences in the conformational dynamics of 6mG residues in G-quadruplex DNAs.
Affiliation: Department of Molecular and Cellular Biochemistry, Center for Structural Biology, University of Kentucky, Lexington, KY 40536, USA.Show MeSH
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Mentions: To discover whether K+-dependent DNA folding affects AGT interaction, binding densities were evaluated at sedimentation equilibrium under conditions of protein excess (Figure 5). Parallel experiments were conducted in TE buffer containing 75 mM KCl and in K+-free buffer, using the unmodified sequence 22wtx and the 6mG-containing sequence G5x (Table 1). Because wild-type AGT rapidly converts O6-methylguanine to unmodified guanine (3,15), the catalytically-inactive C145A mutant AGT was used for these experiments. This protein binds unmodified linear DNAs with stoichiometries and affinities that are indistinguishable from those of the wild-type enzyme (15,27). Data were fit using Equation (2), which embodies a model of binding saturation in which the dominant species are free protein and a protein–DNA complex. High-quality fits with small, randomly-distributed residuals show that this model is consistent with the data. Global analyses of datasets obtained at 3 rotor speeds and two dilutions of each protein–DNA mixture returned stoichiometry values of 1.96 ± 0.14 AGT/22wtx DNA and 1.82 ± 0.21 AGT/G5x DNA, in K+-containing buffer. Parallel analyses carried out in K+-free buffer returned values of 4.12 ± 0.27 AGT/22wtx DNA and 4.38 ± 0.16 AGT/G5x DNA in good agreement with results obtained with single-stranded DNAs of similar length but different sequence (13). Together, these findings suggest that DNA folding to form the G-quadruplex reduces the number of binding sites available to AGT.
Affiliation: Department of Molecular and Cellular Biochemistry, Center for Structural Biology, University of Kentucky, Lexington, KY 40536, USA.