PMID:11907039

Citation	Wong, I, Lundquist, AJ, Bernards, AS and Mosbaugh, DW (2002) Presteady-state analysis of a single catalytic turnover by Escherichia coli uracil-DNA glycosylase reveals a "pinch-pull-push" mechanism. J. Biol. Chem. 277:19424-32
Abstract	Uracil-DNA glycosylase catalyzes the excision of uracils from DNA via a mechanism where the uracil is extrahelically flipped out of the DNA helix into the enzyme active site. A conserved leucine is inserted into the DNA duplex space vacated by the uracil leading to the paradigmatic "push-pull" mechanism of nucleotide flipping. However, the order of these two steps during catalysis has not been conclusively established. We report a complete kinetic analysis of a single catalytic turnover using a hydrolyzable duplex oligodeoxyribonucleotide substrate containing a uracil:2-aminopurine base pair. Rapid chemical-quenched-flow methods defined the kinetics of excision at the active site during catalysis. Stopped-flow fluorometry monitoring the 2-aminopurine fluorescence defined the kinetics of uracil flipping. Parallel experiments detecting the protein fluorescence showed a slower Leu(191) insertion step occurring after nucleotide flipping but before excision. The inserted Leu(191) acts as a doorstop to prevent the return of the flipped-out uracil residue, thereby facilitating the capture of the uracil in the active site and does not play a direct role in "pushing" the uracil out of the DNA helix. The results define for the first time the proper sequence of events during a catalytic cycle and establish a "pull-push", as opposed to a "push-pull", mechanism for nucleotide flipping.
Links	PubMed Online version:10.1074/jbc.M201198200
Keywords	Binding Sites; Catalysis; Catalytic Domain; DNA Glycosylases; Dose-Response Relationship, Drug; Escherichia coli/enzymology; Kinetics; Leucine/chemistry; N-Glycosyl Hydrolases/chemistry; N-Glycosyl Hydrolases/metabolism; Oligodeoxyribonucleotides/chemistry; Protein Conformation; Spectrometry, Fluorescence; Time Factors; Uracil/chemistry; Uracil-DNA Glycosidase
edit table

Significance

You can help EcoliWiki by summarizing why this paper is useful

Useful Materials and Methods

You can help Ecoliwiki by describing the useful materials (strains, plasmids, antibodies, etc) described in this paper.

Annotations

<annotationlinks/>

EcoliWiki Links

Add links to pages that link here (e.g. gene, product, method pages)

References

See Help:References for how to manage references in EcoliWiki.