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

Time course of duplex DNA repair detected by NarI sensitivity. Inset. The 6mG-containing 24-mer duplex [0.037 μM, in 40 mM Tris–acetate (pH 7.9 at 20°C), 100 mM potassium acetate, 2 mM magnesium acetate, 2 mM DTT] mixed with AGT (final concentration 0.074 μM). Aliquots were withdrawn and quenched in 0.2% SDS after 15, 30, 45, 60, 120, 180, 270 and 360s, (samples b–i, respectively); unrepaired DNA is shown in sample a. DNA samples were deproteinized by phenol and ether extractions (28) and then digested with NarI endonuclease. Products were resolved by electrophoresis on a 20% gel. The singly-32P-labeled substrate duplex contains 24 bp and the 32P-labeled digestion fragment 12 bp; band designations give the lengths of the corresponding DNAs. Graph. Time profiles for two repair reactions carried out as described above. The smooth curve is a fit of Equation (4) to the combined data.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 9: Time course of duplex DNA repair detected by NarI sensitivity. Inset. The 6mG-containing 24-mer duplex [0.037 μM, in 40 mM Tris–acetate (pH 7.9 at 20°C), 100 mM potassium acetate, 2 mM magnesium acetate, 2 mM DTT] mixed with AGT (final concentration 0.074 μM). Aliquots were withdrawn and quenched in 0.2% SDS after 15, 30, 45, 60, 120, 180, 270 and 360s, (samples b–i, respectively); unrepaired DNA is shown in sample a. DNA samples were deproteinized by phenol and ether extractions (28) and then digested with NarI endonuclease. Products were resolved by electrophoresis on a 20% gel. The singly-32P-labeled substrate duplex contains 24 bp and the 32P-labeled digestion fragment 12 bp; band designations give the lengths of the corresponding DNAs. Graph. Time profiles for two repair reactions carried out as described above. The smooth curve is a fit of Equation (4) to the combined data.

Mentions: For comparison, we measured repair kinetics for a 6mG-containing 24-mer duplex (oligo sequences given in Table 1), using a NarI cleavage-susceptibility assay (28). This approach was necessary because we could not easily resolve the 6mG-carrying double-stranded DNA from that containing normal guanine, using standard electrophoresis methods (result not shown). NarI endonuclease is inactive against duplex DNA containing a 6mG residue at position 2 in its cognate sequence (42), but repair by AGT restores quantitative NarI cleavage. Shown in Figure 9 are gel data for the time-dependent repair of the 6mG-NarI 24-mer; the graph gives time course data for the experiment shown and for a second, parallel trial. The smooth curve is a fit of Equation (4) to the combined data, showing that a two-phase model is consistent with the kinetics of duplex DNA repair.


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)

Time course of duplex DNA repair detected by NarI sensitivity. Inset. The 6mG-containing 24-mer duplex [0.037 μM, in 40 mM Tris–acetate (pH 7.9 at 20°C), 100 mM potassium acetate, 2 mM magnesium acetate, 2 mM DTT] mixed with AGT (final concentration 0.074 μM). Aliquots were withdrawn and quenched in 0.2% SDS after 15, 30, 45, 60, 120, 180, 270 and 360s, (samples b–i, respectively); unrepaired DNA is shown in sample a. DNA samples were deproteinized by phenol and ether extractions (28) and then digested with NarI endonuclease. Products were resolved by electrophoresis on a 20% gel. The singly-32P-labeled substrate duplex contains 24 bp and the 32P-labeled digestion fragment 12 bp; band designations give the lengths of the corresponding DNAs. Graph. Time profiles for two repair reactions carried out as described above. The smooth curve is a fit of Equation (4) to the combined data.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 9: Time course of duplex DNA repair detected by NarI sensitivity. Inset. The 6mG-containing 24-mer duplex [0.037 μM, in 40 mM Tris–acetate (pH 7.9 at 20°C), 100 mM potassium acetate, 2 mM magnesium acetate, 2 mM DTT] mixed with AGT (final concentration 0.074 μM). Aliquots were withdrawn and quenched in 0.2% SDS after 15, 30, 45, 60, 120, 180, 270 and 360s, (samples b–i, respectively); unrepaired DNA is shown in sample a. DNA samples were deproteinized by phenol and ether extractions (28) and then digested with NarI endonuclease. Products were resolved by electrophoresis on a 20% gel. The singly-32P-labeled substrate duplex contains 24 bp and the 32P-labeled digestion fragment 12 bp; band designations give the lengths of the corresponding DNAs. Graph. Time profiles for two repair reactions carried out as described above. The smooth curve is a fit of Equation (4) to the combined data.
Mentions: For comparison, we measured repair kinetics for a 6mG-containing 24-mer duplex (oligo sequences given in Table 1), using a NarI cleavage-susceptibility assay (28). This approach was necessary because we could not easily resolve the 6mG-carrying double-stranded DNA from that containing normal guanine, using standard electrophoresis methods (result not shown). NarI endonuclease is inactive against duplex DNA containing a 6mG residue at position 2 in its cognate sequence (42), but repair by AGT restores quantitative NarI cleavage. Shown in Figure 9 are gel data for the time-dependent repair of the 6mG-NarI 24-mer; the graph gives time course data for the experiment shown and for a second, parallel trial. The smooth curve is a fit of Equation (4) to the combined data, showing that a two-phase model is consistent with the kinetics of duplex DNA repair.

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