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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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Sedimentation coefficient distributions for telomere sequence and single-stranded DNAs. Sedimentation velocity data were measured at 4°C and 40 000 rpm in 10 mM Tris–HCl (pH 8.0), 1 mM EDTA supplemented with 75 mM KCl and 5 mM MgCl2. DNA concentrations were ∼0.9 μM (A260 ∼0.25). Smooth curves are c(S) distributions calculated with the program Sedfit (30,35).
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Figure 2: Sedimentation coefficient distributions for telomere sequence and single-stranded DNAs. Sedimentation velocity data were measured at 4°C and 40 000 rpm in 10 mM Tris–HCl (pH 8.0), 1 mM EDTA supplemented with 75 mM KCl and 5 mM MgCl2. DNA concentrations were ∼0.9 μM (A260 ∼0.25). Smooth curves are c(S) distributions calculated with the program Sedfit (30,35).

Mentions: Sedimentation velocity experiments were performed to determine whether the telomere-sequence DNAs containing 6mG modifications formed compact structures. Dissolved in TE buffer containing 75 mM potassium chloride, both unmodified and 6mG variants of the telomere sequence sedimented as single species with s20,w ∼2.0 (Figure 2A, Table 2). These values are similar to one previously reported for the quadruplex form of the unmodified 22-mer (22wt) (24). Molecular weight (c(M)) distributions (35) for the 22wt DNA and for sequences containing 6mG at positions G1, G3, G4 or G6 (outer quartet locations) returned mean values (7850 < Mr < 8760), slightly greater than the sequence molecular weights of the DNAs (Mr ∼ 7600), supporting the notion that these species were primarily monomeric (Tables 1 and 2). However, DNAs with 6mG modifications at inner-quartet positions (G2, G5 and G11) gave higher apparent molecular weights (9695 < Mr < 10 938) suggesting the presence of a modest mole-fraction of higher oligomeric species. The sedimentation coefficient (c(S)) distributions for these DNAs were also broader than those of the others (Figure 2), consistent with models in which monomers and multimers exchange rapidly during sedimentation.


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)

Sedimentation coefficient distributions for telomere sequence and single-stranded DNAs. Sedimentation velocity data were measured at 4°C and 40 000 rpm in 10 mM Tris–HCl (pH 8.0), 1 mM EDTA supplemented with 75 mM KCl and 5 mM MgCl2. DNA concentrations were ∼0.9 μM (A260 ∼0.25). Smooth curves are c(S) distributions calculated with the program Sedfit (30,35).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: Sedimentation coefficient distributions for telomere sequence and single-stranded DNAs. Sedimentation velocity data were measured at 4°C and 40 000 rpm in 10 mM Tris–HCl (pH 8.0), 1 mM EDTA supplemented with 75 mM KCl and 5 mM MgCl2. DNA concentrations were ∼0.9 μM (A260 ∼0.25). Smooth curves are c(S) distributions calculated with the program Sedfit (30,35).
Mentions: Sedimentation velocity experiments were performed to determine whether the telomere-sequence DNAs containing 6mG modifications formed compact structures. Dissolved in TE buffer containing 75 mM potassium chloride, both unmodified and 6mG variants of the telomere sequence sedimented as single species with s20,w ∼2.0 (Figure 2A, Table 2). These values are similar to one previously reported for the quadruplex form of the unmodified 22-mer (22wt) (24). Molecular weight (c(M)) distributions (35) for the 22wt DNA and for sequences containing 6mG at positions G1, G3, G4 or G6 (outer quartet locations) returned mean values (7850 < Mr < 8760), slightly greater than the sequence molecular weights of the DNAs (Mr ∼ 7600), supporting the notion that these species were primarily monomeric (Tables 1 and 2). However, DNAs with 6mG modifications at inner-quartet positions (G2, G5 and G11) gave higher apparent molecular weights (9695 < Mr < 10 938) suggesting the presence of a modest mole-fraction of higher oligomeric species. The sedimentation coefficient (c(S)) distributions for these DNAs were also broader than those of the others (Figure 2), consistent with models in which monomers and multimers exchange rapidly during sedimentation.

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