Limits...
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.

Show MeSH

Related in: MedlinePlus

Circular dichroism spectra of telomere-sequence DNAs. (A) The 22wt and telomere sequences containing 6mG at outer tetrad positions G1 or G3 or G4 or G6, as indicated in the legend. Spectra were taken at 10°C in buffer containing 10 mM Tris–HCl (pH 8.0), 1 mM EDTA, 75 mM KCl and 5 mM MgCl2. (B) Spectra of telomere sequences containing 6mG at inner tetrad positions G2, G5 or G11, as indicated in the legend. Solution conditions were the same as in (A). (C) Spectra of 22wt and G5 sequences taken at 10°C in 10 mM Tris–HCl (pH 8.0), 1 mM EDTA (TE buffer) either alone or supplemented with 75 mM KCl or with 75 mM TEA, as indicated in the legend. (D) Comparison of spectra for the 22-mer G11 and 25-mer G11 DNAs. Spectra were taken at 10°C in 10 mM Tris–HCl (pH 8.0), 1 mM EDTA (TE buffer) and 75 mM KCl. DNA concentrations were 5 μM in all measurements.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4150771&req=5

Figure 3: Circular dichroism spectra of telomere-sequence DNAs. (A) The 22wt and telomere sequences containing 6mG at outer tetrad positions G1 or G3 or G4 or G6, as indicated in the legend. Spectra were taken at 10°C in buffer containing 10 mM Tris–HCl (pH 8.0), 1 mM EDTA, 75 mM KCl and 5 mM MgCl2. (B) Spectra of telomere sequences containing 6mG at inner tetrad positions G2, G5 or G11, as indicated in the legend. Solution conditions were the same as in (A). (C) Spectra of 22wt and G5 sequences taken at 10°C in 10 mM Tris–HCl (pH 8.0), 1 mM EDTA (TE buffer) either alone or supplemented with 75 mM KCl or with 75 mM TEA, as indicated in the legend. (D) Comparison of spectra for the 22-mer G11 and 25-mer G11 DNAs. Spectra were taken at 10°C in 10 mM Tris–HCl (pH 8.0), 1 mM EDTA (TE buffer) and 75 mM KCl. DNA concentrations were 5 μM in all measurements.

Mentions: Circular dichroism (CD) spectroscopy was used to further characterize the folding of these DNAs. In TE + 75 mM KCl buffer, the unmodified 22wt DNA and sequences with 6mG at residues G1, G3, G4 and G6 had CD maxima near 295 nm and minima near 235 nm (Figure 3A). These features correspond well to spectra previously observed for antiparallel strand quadruplex DNA (38). They also resemble previously-determined spectra of a folded form of the human sequence (24). In contrast, spectra for telomere sequences with 6mG at positions G2, G5 and G11 have distinct maxima near 260 nm (Figure 3B); maxima in this wavelength range have been described for parallel G-quadruplex DNAs (38,39). Finally, the CD spectra of 22wtx and G5x DNAs in K+-free buffers differ significantly from those obtained in the presence of K+, indicating the presence of conformations that are distinct from the quadruplex fold (Figure 3C). Spectra obtained in TE buffer and TE buffer containing 75mM TEA–HCl were similar, indicating that the corresponding conformations are not unique to low [salt] conditions.


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 of telomere-sequence DNAs. (A) The 22wt and telomere sequences containing 6mG at outer tetrad positions G1 or G3 or G4 or G6, as indicated in the legend. Spectra were taken at 10°C in buffer containing 10 mM Tris–HCl (pH 8.0), 1 mM EDTA, 75 mM KCl and 5 mM MgCl2. (B) Spectra of telomere sequences containing 6mG at inner tetrad positions G2, G5 or G11, as indicated in the legend. Solution conditions were the same as in (A). (C) Spectra of 22wt and G5 sequences taken at 10°C in 10 mM Tris–HCl (pH 8.0), 1 mM EDTA (TE buffer) either alone or supplemented with 75 mM KCl or with 75 mM TEA, as indicated in the legend. (D) Comparison of spectra for the 22-mer G11 and 25-mer G11 DNAs. Spectra were taken at 10°C in 10 mM Tris–HCl (pH 8.0), 1 mM EDTA (TE buffer) and 75 mM KCl. DNA concentrations were 5 μM in all measurements.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150771&req=5

Figure 3: Circular dichroism spectra of telomere-sequence DNAs. (A) The 22wt and telomere sequences containing 6mG at outer tetrad positions G1 or G3 or G4 or G6, as indicated in the legend. Spectra were taken at 10°C in buffer containing 10 mM Tris–HCl (pH 8.0), 1 mM EDTA, 75 mM KCl and 5 mM MgCl2. (B) Spectra of telomere sequences containing 6mG at inner tetrad positions G2, G5 or G11, as indicated in the legend. Solution conditions were the same as in (A). (C) Spectra of 22wt and G5 sequences taken at 10°C in 10 mM Tris–HCl (pH 8.0), 1 mM EDTA (TE buffer) either alone or supplemented with 75 mM KCl or with 75 mM TEA, as indicated in the legend. (D) Comparison of spectra for the 22-mer G11 and 25-mer G11 DNAs. Spectra were taken at 10°C in 10 mM Tris–HCl (pH 8.0), 1 mM EDTA (TE buffer) and 75 mM KCl. DNA concentrations were 5 μM in all measurements.
Mentions: Circular dichroism (CD) spectroscopy was used to further characterize the folding of these DNAs. In TE + 75 mM KCl buffer, the unmodified 22wt DNA and sequences with 6mG at residues G1, G3, G4 and G6 had CD maxima near 295 nm and minima near 235 nm (Figure 3A). These features correspond well to spectra previously observed for antiparallel strand quadruplex DNA (38). They also resemble previously-determined spectra of a folded form of the human sequence (24). In contrast, spectra for telomere sequences with 6mG at positions G2, G5 and G11 have distinct maxima near 260 nm (Figure 3B); maxima in this wavelength range have been described for parallel G-quadruplex DNAs (38,39). Finally, the CD spectra of 22wtx and G5x DNAs in K+-free buffers differ significantly from those obtained in the presence of K+, indicating the presence of conformations that are distinct from the quadruplex fold (Figure 3C). Spectra obtained in TE buffer and TE buffer containing 75mM TEA–HCl were similar, indicating that the corresponding conformations are not unique to low [salt] conditions.

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