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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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Thermal melting of G-quadruplex DNAs detected by circular dichroism. Samples were annealed at 37°C in 10 mM Tris–HCl (pH 7.6), 1 mM EDTA, 75 mM KCl, cooled slowly to 20°C for handling and then equilibrated at 0°C for 10 min prior to the start of analysis. Temperature was increased at 1°C min−1 with dichroism amplitudes measured at 295 nm, every 0.5°C.
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Figure 4: Thermal melting of G-quadruplex DNAs detected by circular dichroism. Samples were annealed at 37°C in 10 mM Tris–HCl (pH 7.6), 1 mM EDTA, 75 mM KCl, cooled slowly to 20°C for handling and then equilibrated at 0°C for 10 min prior to the start of analysis. Temperature was increased at 1°C min−1 with dichroism amplitudes measured at 295 nm, every 0.5°C.

Mentions: Thermal denaturation profiles were obtained by monitoring CD changes at 295 nm (Figure 4). Under these buffer conditions, all DNAs had well-defined thermal transitions with low-temperature baselines that extended to ∼293°K, allowing sample handling at just-below room temperature without risk of denaturation. Transition midpoint (Tm) temperatures ranged from 337°K (22wt DNA) to 313°K (G5 DNA), in the order 22wt > G1 > G6 > G4 > G3 > G11 > G2 > G5. Thus, folded forms of 6mG-containing DNAs had lower thermal stabilities than that of the unmodified DNA, and folded DNAs with 6mG residues in outer tetrads (G1, G3, G4, G6) were more stable than those with 6mG residues in inner tetrads (G2, G5, G11). This trend is consistent with and extends earlier observations made with telomere-sequence DNAs containing 6mG at positions G1, G2 and G3 (24). Together these hydrodynamic, spectroscopic and melting results are consistent with models in which these DNAs undergo K+-dependent folding to attain compact quadruplex conformations.


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)

Thermal melting of G-quadruplex DNAs detected by circular dichroism. Samples were annealed at 37°C in 10 mM Tris–HCl (pH 7.6), 1 mM EDTA, 75 mM KCl, cooled slowly to 20°C for handling and then equilibrated at 0°C for 10 min prior to the start of analysis. Temperature was increased at 1°C min−1 with dichroism amplitudes measured at 295 nm, every 0.5°C.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: Thermal melting of G-quadruplex DNAs detected by circular dichroism. Samples were annealed at 37°C in 10 mM Tris–HCl (pH 7.6), 1 mM EDTA, 75 mM KCl, cooled slowly to 20°C for handling and then equilibrated at 0°C for 10 min prior to the start of analysis. Temperature was increased at 1°C min−1 with dichroism amplitudes measured at 295 nm, every 0.5°C.
Mentions: Thermal denaturation profiles were obtained by monitoring CD changes at 295 nm (Figure 4). Under these buffer conditions, all DNAs had well-defined thermal transitions with low-temperature baselines that extended to ∼293°K, allowing sample handling at just-below room temperature without risk of denaturation. Transition midpoint (Tm) temperatures ranged from 337°K (22wt DNA) to 313°K (G5 DNA), in the order 22wt > G1 > G6 > G4 > G3 > G11 > G2 > G5. Thus, folded forms of 6mG-containing DNAs had lower thermal stabilities than that of the unmodified DNA, and folded DNAs with 6mG residues in outer tetrads (G1, G3, G4, G6) were more stable than those with 6mG residues in inner tetrads (G2, G5, G11). This trend is consistent with and extends earlier observations made with telomere-sequence DNAs containing 6mG at positions G1, G2 and G3 (24). Together these hydrodynamic, spectroscopic and melting results are consistent with models in which these DNAs undergo K+-dependent folding to attain compact quadruplex conformations.

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