Repair of O6-methylguanine adducts in human telomeric G-quadruplex DNA by O6-alkylguanine-DNA alkyltransferase.
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.
Affiliation: Department of Molecular and Cellular Biochemistry, Center for Structural Biology, University of Kentucky, Lexington, KY 40536, USA.Show MeSH
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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.
Affiliation: Department of Molecular and Cellular Biochemistry, Center for Structural Biology, University of Kentucky, Lexington, KY 40536, USA.