Presteady-state Analysis of a Single Catalytic Turnover by Escherichia coli Uracil-DNA Glycosylase Reveals a “Pinch-Pull-Push” Mechanism

Wong, Isaac; Lundquist, Amy J.; Bernards, Andrew S.; Mosbaugh, Dale W.

doi:10.1074/jbc.m201198200

Cited by 71 publications

(77 citation statements)

References 47 publications

(49 reference statements)

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“…When analysing kinetic parameters of E. coli Ung L191A against single-stranded substrates, a 15-fold reduction in k cat /K m was observed compared to the wild type (Handa et al, 2001;Jiang and Stivers, 2001), which would not be expected in the absence of stabilizing base pairing. A likely explanation for this could be that the leucine side chain rather functions as a 'doorstop' to prevent return of the flipped out uracil residue, as recently suggested by Wong et al (2002). Using stopped-flow experiments of E. coli Ung and monitoring fluorescence of 2-aminopurine adjacent to uracil, they demonstrated that Leu191 insertion occurred after nucleotide-flipping but before excision, thus indicating a 'pinch -pull -push' rather than a 'pinch -push -pull' mechanism.…”

Section: The Catalytic Domain Of Ung Proteinsmentioning

confidence: 99%

Uracil in DNA – occurrence, consequences and repair

2002

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Section: The Catalytic Domain Of Ung Proteinsmentioning

confidence: 99%

Uracil in DNA – occurrence, consequences and repair

2002

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“…First, the structural information on cognate and nonspecific complexes suggests that loop positioning is determined by the sequence of the bound DNA, thus providing a molecular basis for an inducedfit mechanism. Second, the temporal assignment of loop movement in relation to other known steps in the reaction cycle, such as base flipping, could be insightful for understanding if these processes are enzyme-assisted or occur passively (9,34). Third, our recent work on M.HhaI and other DNA methyltransferases suggests that conformational rearrangements such as the loop movement in M.HhaI or DNA bending by M.EcoRI can directly contribute to specificity (29,30,35).…”

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Observing an Induced-fit Mechanism during Sequence-specific DNA Methylation

Estabrook

Reich

2006

Journal of Biological Chemistry

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The characterization of conformational changes that drive induced-fit mechanisms and their quantitative importance to enzyme specificity are essential for a full understanding of enzyme function. Here, we report on M.HhaI, a sequence-specific DNA cytosine C 5 methyltransferase that reorganizes a flexible loop (residues 80 -100) upon binding cognate DNA as part of an induced-fit mechanism. To directly observe this ϳ26 Å conformational rearrangement and provide a basis for understanding its importance to specificity, we replaced loop residues Lys-91 and Glu-94 with tryptophans. The double mutants W41F/K91W and W41F/E94W are relatively unperturbed in kinetic and thermodynamic properties. Ligand-induced changes in protein conformation can contribute to enzyme catalysis, regulation of function, and substrate specificity. Induced-fit mechanisms involve conformational rearrangements of an enzyme to facilitate or enhance correct substrate binding and can provide improved specificity (1-3). First introduced in 1958 (4), induced-fit mechanisms contribute to diverse biological processes (5), including tRNA binding in ribosomes (6), DNA-modifying enzymes (7-9), kinases (10, 11), RNA folding (12), DNA binding specificity (13,14), polymerases (15, 16), and many others. However, direct evidence for an induced-fit mechanism through the observation of real-time protein conformational rearrangements coupled to specificity have been quantitated for a small number of enzymes (17)(18)(19)(20).Enzymes that sequence-specifically modify DNA, including nucleases, repair enzymes, and methyltransferases are faced with severe challenges of substrate recognition and specificity due to the overwhelming abundance of sites that are closely related to the cognate sequence (1,3,5,21). Mechanisms posited to account for this discrimination are diverse and often require an induced-fit process as the enzyme-DNA complex moves from a nonspecific site to the cognate sequence (22, 23). The availability of high resolution structures of cognate DNAenzyme complexes for many such systems provides a detailed understanding of specific interactions leading to tight and cognate binding (24 -27). However, interactions leading to nonspecific substrate binding, which facilitate site searching, are far less characterized (1, 3, 28). Furthermore, the interconversion of conformers as the enzyme goes from nonspecific to cognate sites prior to forming the catalytically competent complex can contribute to such specificity (13, 21, 28 -31).The DNA cytosine C 5 methyltransferase M.HhaI binds DNA substrates between its two domains and the cofactor AdoMet 2 in the large domain near the active site (see Fig. 1). Inspection of two cocrystal structures of M.HhaI, one involving the cognate DNA and the cofactor product AdoHcy (3MHT.pdb) (24), the other involving nonspecific DNA and AdoHcy (2HMY.pdb) (32), suggest that the enzyme may exploit an induced-fit mechanism. Induced-fit DNA binding was first proposed for M.HhaI in 1994 when the first ternary complex with cognate DNA, cof...

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“…The amplitude of the burst phase, therefore, provided a direct measure of the molar amount of productively bound substrate during the initial turnover (45)(46)(47). In active site titrations, burst amplitudes were measure at different MutY and DNA concentrations.…”

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A Dimeric Mechanism for Contextual Target Recognition by MutY Glycosylase

Wong

Bernards

Miller

et al. 2003

Journal of Biological Chemistry

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MutY, an adenine glycosylase, initiates the critical repair of an adenine:8-oxo-guanine base pair in DNA arising from polymerase error at the oxidatively damaged guanine. Here we demonstrate for the first time, using presteady-state active site titrations, that MutY assembles into a dimer upon binding substrate DNA and that the dimer is the functionally active form of the enzyme. Additionally, we observed allosteric inhibition of glycosylase activity in the dimer by the concurrent binding of two lesion mispairs. Active site titration results were independently verified by gel mobility shift assays and quantitative DNA footprint titrations. A model is proposed for the potential functional role of the observed polysteric and allosteric regulation in recruiting and coordinating interactions with the methyl-directed mismatch repair system.

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Presteady-state Analysis of a Single Catalytic Turnover by Escherichia coli Uracil-DNA Glycosylase Reveals a “Pinch-Pull-Push” Mechanism

Cited by 71 publications

References 47 publications

Uracil in DNA – occurrence, consequences and repair

Uracil in DNA – occurrence, consequences and repair

Observing an Induced-fit Mechanism during Sequence-specific DNA Methylation

A Dimeric Mechanism for Contextual Target Recognition by MutY Glycosylase

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