Dataset Information

Role of two strictly conserved residues in nucleotide flipping and N-glycosylic bond cleavage by human thymine DNA glycosylase.

ABSTRACT: Thymine DNA glycosylase (TDG) promotes genomic integrity by excising thymine from mutagenic G.T mismatches arising by deamination of 5-methylcytosine, and follow-on base excision repair enzymes restore a G.C pair. TDG cleaves the N-glycosylic bond of dT and some other nucleotides, including 5-substituted 2'-deoxyuridine analogs, once they have been flipped from the helix into its active site. We examined the role of two strictly conserved residues; Asn(140), implicated in the chemical step, and Arg(275), implicated in nucleotide flipping. The N140A variant binds substrate DNA with the same tight affinity as wild-type TDG, but it has no detectable base excision activity for a G.T substrate, and its excision rate is vastly diminished (by approximately 10(4.4)-fold) for G.U, G.FU, and G.BrU substrates. Thus, Asn(140) does not contribute substantially to substrate binding but is essential for the chemical step, where it stabilizes the transition state by approximately 6 kcal/mol (compared with 11.6 kcal/mol stabilization provided by TDG overall). Our recent crystal structure revealed that Arg(275) penetrates the DNA minor groove, filling the void created by nucleotide flipping. We found that the R275A and R275L substitutions weaken substrate binding and substantially decrease the base excision rate for G.T and G.BrU substrates. Our results indicate that Arg(275) promotes and/or stabilizes nucleotide flipping, a role that is most important for target nucleotides that are relatively large (dT and bromodeoxyuridine) and/or have a stable N-glycosylic bond (dT). Arg(275) does not contribute substantially to the binding of TDG to abasic DNA product, and it cannot account for the slow product release exhibited by TDG.

SUBMITTER: Maiti A

PROVIDER: S-EPMC2794782 | biostudies-literature | 2009 Dec

REPOSITORIES: biostudies-literature

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Publications

Role of two strictly conserved residues in nucleotide flipping and N-glycosylic bond cleavage by human thymine DNA glycosylase.

Maiti Atanu A Morgan Michael T MT Drohat Alexander C AC

The Journal of biological chemistry 20091030 52

Thymine DNA glycosylase (TDG) promotes genomic integrity by excising thymine from mutagenic G.T mismatches arising by deamination of 5-methylcytosine, and follow-on base excision repair enzymes restore a G.C pair. TDG cleaves the N-glycosylic bond of dT and some other nucleotides, including 5-substituted 2'-deoxyuridine analogs, once they have been flipped from the helix into its active site. We examined the role of two strictly conserved residues; Asn(140), implicated in the chemical step, and ...[more]

PMID: 19880517

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Project description:Human alkyladenine DNA glycosylase (AAG) locates and excises a wide variety of structurally diverse alkylated and oxidized purine lesions from DNA to initiate the base excision repair pathway. Recognition of a base lesion requires flipping of the damaged nucleotide into a relatively open active site pocket between two conserved tyrosine residues, Y127 and Y159. We have mutated each of these amino acids to tryptophan and measured the kinetic effects on the nucleotide flipping and base excision steps. The Y127W and Y159W mutant proteins have robust glycosylase activity toward DNA containing 1,N(6)-ethenoadenine (?A), within 4-fold of that of the wild-type enzyme, raising the possibility that tryptophan fluorescence could be used to probe the DNA binding and nucleotide flipping steps. Stopped-flow fluorescence was used to compare the time-dependent changes in tryptophan fluorescence and ?A fluorescence. For both mutants, the tryptophan fluorescence exhibited two-step binding with essentially identical rate constants as were observed for the ?A fluorescence changes. These results provide evidence that AAG forms an initial recognition complex in which the active site pocket is perturbed and the stacking of the damaged base is disrupted. Upon complete nucleotide flipping, there is further quenching of the tryptophan fluorescence with coincident quenching of the ?A fluorescence. Although these mutations do not have large effects on the rate constant for excision of ?A, there are dramatic effects on the rate constants for nucleotide flipping that result in 40-100-fold decreases in the flipping equilibrium relative to wild-type. Most of this effect is due to an increased rate of unflipping, but surprisingly the Y159W mutation causes a 5-fold increase in the rate constant for flipping. The large effect on the equilibrium for nucleotide flipping explains the greater deleterious effects that these mutations have on the glycosylase activity toward base lesions that are in more stable base pairs.

Dataset Information

Role of two strictly conserved residues in nucleotide flipping and N-glycosylic bond cleavage by human thymine DNA glycosylase.

Publications

Role of two strictly conserved residues in nucleotide flipping and N-glycosylic bond cleavage by human thymine DNA glycosylase.

Similar Datasets

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