Presteady-state analysis of a single catalytic turnover by Escherichia coli uracil-DNA glycosylase reveals a "pinch-pull-push" mechanism

The Journal of Biological Chemistry
Isaac WongDale W Mosbaugh

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...Continue Reading

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