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Repair of O6-methylguanine adducts in human telomeric G-quadruplex DNA by O6-alkylguanine-DNA alkyltransferase.

Hellman LM, Spear TJ, Koontz CJ, Melikishvili M, Fried MG - Nucleic Acids Res. (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biochemistry, Center for Structural Biology, University of Kentucky, Lexington, KY 40536, USA.

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Circular dichroism spectra and difference spectra of AGT–DNA mixtures. (A) Spectrum of 22wt DNA (solid line) and difference spectrum (spectrum of AGT-22wt DNA mixture minus that of AGT alone; dashed line). The 22wt DNA and C145A-AGT concentrations were 5 and 50 μM, respectively, in buffer consisting of 10 mM Tris–HCl (pH 8.0), 1 mM EDTA, 75 mM KCl and 5 mM MgCl2. (B) Spectrum of the single-stranded 26-mer alone (solid line) and difference spectrum (spectrum of AGT-26-mer DNA mixture minus that of free AGT; dashed line). The 26-mer DNA and C145A-AGT concentrations were 3 and 30 μM, respectively. All spectra were recorded at 4°C.
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Figure 6: Circular dichroism spectra and difference spectra of AGT–DNA mixtures. (A) Spectrum of 22wt DNA (solid line) and difference spectrum (spectrum of AGT-22wt DNA mixture minus that of AGT alone; dashed line). The 22wt DNA and C145A-AGT concentrations were 5 and 50 μM, respectively, in buffer consisting of 10 mM Tris–HCl (pH 8.0), 1 mM EDTA, 75 mM KCl and 5 mM MgCl2. (B) Spectrum of the single-stranded 26-mer alone (solid line) and difference spectrum (spectrum of AGT-26-mer DNA mixture minus that of free AGT; dashed line). The 26-mer DNA and C145A-AGT concentrations were 3 and 30 μM, respectively. All spectra were recorded at 4°C.

Mentions: We used CD spectroscopy to test whether AGT binding alters the secondary structures of our quadruplex DNAs. Difference spectra (CDDNA+AGT – CDAGT) were obtained for the 22wt quadruplex in TE buffer containing 75 mM KCl and compared to spectra obtained at the same [DNA], but in the absence of AGT. As shown in Figure 6A, inclusion of AGT in the solution does not significantly change the DNA spectrum. This contrasts with the effect of AGT addition to a solution of the single-stranded 26-nt DNA, which results in a significant CD difference throughout the near-ultraviolet range (Figure 6B). These outcomes are simply explained by models in which AGT binding causes a substantial conformational shift in single-stranded 26-mer DNA, but not in the 22wt G-quadruplex.


Repair of O6-methylguanine adducts in human telomeric G-quadruplex DNA by O6-alkylguanine-DNA alkyltransferase.

Hellman LM, Spear TJ, Koontz CJ, Melikishvili M, Fried MG - Nucleic Acids Res. (2014)

Circular dichroism spectra and difference spectra of AGT–DNA mixtures. (A) Spectrum of 22wt DNA (solid line) and difference spectrum (spectrum of AGT-22wt DNA mixture minus that of AGT alone; dashed line). The 22wt DNA and C145A-AGT concentrations were 5 and 50 μM, respectively, in buffer consisting of 10 mM Tris–HCl (pH 8.0), 1 mM EDTA, 75 mM KCl and 5 mM MgCl2. (B) Spectrum of the single-stranded 26-mer alone (solid line) and difference spectrum (spectrum of AGT-26-mer DNA mixture minus that of free AGT; dashed line). The 26-mer DNA and C145A-AGT concentrations were 3 and 30 μM, respectively. All spectra were recorded at 4°C.
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Related In: Results  -  Collection

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Figure 6: Circular dichroism spectra and difference spectra of AGT–DNA mixtures. (A) Spectrum of 22wt DNA (solid line) and difference spectrum (spectrum of AGT-22wt DNA mixture minus that of AGT alone; dashed line). The 22wt DNA and C145A-AGT concentrations were 5 and 50 μM, respectively, in buffer consisting of 10 mM Tris–HCl (pH 8.0), 1 mM EDTA, 75 mM KCl and 5 mM MgCl2. (B) Spectrum of the single-stranded 26-mer alone (solid line) and difference spectrum (spectrum of AGT-26-mer DNA mixture minus that of free AGT; dashed line). The 26-mer DNA and C145A-AGT concentrations were 3 and 30 μM, respectively. All spectra were recorded at 4°C.
Mentions: We used CD spectroscopy to test whether AGT binding alters the secondary structures of our quadruplex DNAs. Difference spectra (CDDNA+AGT – CDAGT) were obtained for the 22wt quadruplex in TE buffer containing 75 mM KCl and compared to spectra obtained at the same [DNA], but in the absence of AGT. As shown in Figure 6A, inclusion of AGT in the solution does not significantly change the DNA spectrum. This contrasts with the effect of AGT addition to a solution of the single-stranded 26-nt DNA, which results in a significant CD difference throughout the near-ultraviolet range (Figure 6B). These outcomes are simply explained by models in which AGT binding causes a substantial conformational shift in single-stranded 26-mer DNA, but not in the 22wt G-quadruplex.

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biochemistry, Center for Structural Biology, University of Kentucky, Lexington, KY 40536, USA.

Show MeSH
Related in: MedlinePlus