Binding of specific DNA base-pair mismatches by N-methylpurine-DNA glycosylase and its implication in initial damage recognition.
Most DNA glycosylases including N-methylpurine-DNA glycosylase (MPG), which initiate DNA base excision repair, have a wide substrate range of damaged or altered bases in duplex DNA. In contrast, uracil-DNA glycosylase (UDG) is specific for uracil and excises it from both single-stranded and duplex DNAs. Here we show by DNA footprinting analysis that MPG, but not UDG, bound to base-pair mismatches especially to less stable pyrimidine-pyrimidine pairs, without catalyzing detectable base cleavage. Thermal denaturation studies of these normal and damaged (e.g. 1,N(6)-ethenoadenine, varepsilonA) base mispairs indicate that duplex instability rather than exact fit of the flipped out damaged base in the catalytic pocket is a major determinant in the initial recognition of damage by MPG. Finally, based on our determination of binding affinity and catalytic efficiency we conclude that the initial recognition of substrate base lesions by MPG is dependent on the ease of flipping of the base from unstable pairs to a flexible catalytic pocket.
Biswas
Tapan
T
Sealy Center for Molecular Science and Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston 77555-1079, USA.
Clos
Lawrence J
LJ
SantaLucia
John
J
Mitra
Sankar
S
Roy
Rabindra
R
D000818:Animals; D020137:Base Pair Mismatch; D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage; D018983:DNA Footprinting; D045647:DNA Glycosylases; D004260:DNA Repair / Q000502:physiology*; D004355:Drug Stability; D066298:In Vitro Techniques; D051379:Mice; D008954:Models, Biological; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000378:metabolism*; D009691:Nucleic Acid Denaturation; D013379:Substrate Specificity; D051981:Uracil-DNA Glycosidase
D004247:DNA; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase; C076131:DNA-3-methyladenine glycosidase II