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Base-Flipping Mutations of Uracil DNA Glycosylase: Substrate Rescue Using a Pyrene Nucleotide Wedge
- Source :
- Biochemistry. 41:11248-11254
- Publication Year :
- 2002
- Publisher :
- American Chemical Society (ACS), 2002.
-
Abstract
- We recently introduced a new substrate rescue tool for investigating enzymatic base flipping by uracil DNA glycosylase (UDG) in which a bulky pyrene nucleotide wedge (Y) was placed opposite a uracil in duplex DNA (i.e., a U/Y pair), thereby preorganizing the target base in an extrahelical conformation [Jiang, Y. L., et al. (2001) J. Biol. Chem. 276, 42347-54]. The pyrene wedge completely rescued the large catalytic defects resulting from removal of the natural Leu191 wedge, presumably mimicking the pushing and plugging function of this group. Here we employ the pyrene rescue method in combination with transient kinetic approaches to assess the functional roles of six conserved enzymatic groups of UDG that have been implicated in the "pinch, push, plug, and pull" base-flipping mechanism (see the preceding paper in this issue). We find that a U/Y base pair increases the apparent second-order rate constant for damaged site recognition by L191G pushing mutation by 45-fold as compared to a U/A pair, thereby fully rescuing the kinetic effects of the mutation. Remarkably, the U/Y pair also allows L191G to proceed through the conformational docking step that is severely comprised with the normal U/A substrate, and allows the active site of UDG to clamp around the extrahelical base. Thus, pyrene also fulfills the plugging role of the Leu191 side chain. Preorganization of uracil in an extrahelical conformation by pyrene allows diffusion-controlled damage recognition by all of these base-flipping mutants, and allows the UDG conformational change to proceed as rapidly as the rate of uracil flipping with the natural U/A base pair. Thus, the pyrene wedge substrate allows UDG to recognize uracil by a lock-and-key mechanism, rather than the natural induced-fit mechanism. Unnatural pyrene base pairs may provide a general strategy to promote site-specific targeting of other enzymes that recognize extrahelical bases.
- Subjects :
- Conformational change
Base pair
Stereochemistry
Biochemistry
DNA Glycosylases
Substrate Specificity
chemistry.chemical_compound
Organophosphorus Compounds
Nucleotide
Uracil
Uracil-DNA Glycosidase
Base Pairing
N-Glycosyl Hydrolases
chemistry.chemical_classification
Pyrenes
biology
Nucleotides
Chemistry
Nucleic Acid Heteroduplexes
Active site
Kinetics
Spectrometry, Fluorescence
Amino Acid Substitution
DNA glycosylase
Uracil-DNA glycosylase
Mutagenesis, Site-Directed
biology.protein
Pyrene
Protein Binding
Subjects
Details
- ISSN :
- 15204995 and 00062960
- Volume :
- 41
- Database :
- OpenAIRE
- Journal :
- Biochemistry
- Accession number :
- edsair.doi.dedup.....020df929e7132364fc94bb844a550873