Enzymatic capture of an extrahelical thymine in the search for uracil in DNA

Abstract

The enzyme uracil DNA glycosylase (UNG) excises unwanted uracil bases in the genome using an extrahelical base recognition mechanism. Efficient removal of uracil is essential for prevention of C-to-T transition mutations arising from cytosine deamination, cytotoxic U*A pairs arising from incorporation of dUTP in DNA, and for increasing immunoglobulin gene diversity during the acquired immune response. A central event in all of these UNG-mediated processes is the singling out of rare U*A or U*G base pairs in a background of approximately 10(9) T*A or C*G base pairs in the human genome. Here we establish for the human and Escherichia coli enzymes that discrimination of thymine and uracil is initiated by thermally induced opening of T*A and U*A base pairs and not by active participation of the enzyme. Thus, base-pair dynamics has a critical role in the genome-wide search for uracil, and may be involved in initial damage recognition by other DNA repair glycosylases.

A two-step nucleotide-flipping mechanism enables kinetic discrimination of DNA lesions by AGT

“…That the protein significantly stabilizes a base separated state (compare with SI Fig. 8) is consistent with findings for other DNA-repair proteins (4,7,8,10,26). For the active-site entry step, it was necessary to substitute the distance between the O 6 atom of the flipping base and the C ␣ atom of Ala-154 (d 5 ) for d 3 because Gua lacks the methyl carbon.…”

“…Efficient means for generating initial paths (14) together with informatic methods (13) that we introduced now enable us to harvest a statistically significant number of trajectories of a biomedically important stochastic process in its entirety and identify the features that characterize the ensemble of transition states. In contrast to the ''push-pull'' mechanism once proposed for nucleotide flipping (2, 5), we observe a two-step process that promotes a kinetic, rather than a thermodynamic (3,7,8), gate-keeping strategy for lesion discrimination.…”

“…As noted above, there is precedent for extrahelical intermediates in other DNA-repair systems. Base analogs have been used to obtain crystal structures of complexes of UDG and hOGG1 with partially flipped bases (3,8). Although it is possible for base analogs to stabilize off-pathway intermediates, complementary data from NMR imino proton exchange experiments on UDG with a variety of DNA duplexes (7) suggest that these structures are meaningful representations of the unperturbed systems.…”

“…Based on an x-ray crystallographic structure of uracil-DNA glycosylase (UDG) (2), it was proposed that, as the protein compresses the DNA backbone, the nucleotide that flips is ''pushed'' by a finger residue and ''pulled'' by specific interactions in the active site (5,6). Alternatively, more passive mechanisms in which the protein shifts the equilibrium for base pair opening have been suggested (7,8). Evaluating these mechanisms for different proteins in physiologically relevant contexts is important for understanding how nucleotide-flipping proteins identify their targets (4).…”

Early steps in the DNA base excision/single-strand interruption repair pathway in mammalian cells

“…DNA binds along a positively charged groove in the enzyme, but the tight-fitting uracil-binding pocket located at the base of this groove is too deep and narrow to allow binding of DNA-uracil unless it is 'flipped out' of the DNA helix. The structure of human UDG and that bound to uracil-containing oligo has demonstrated the basis for the enzyme-assisted nucleotide flipping [21,22]. Another DNA glycosylase SMUG1 has been characterized 30 npg in human cells [23,24], which catalyzes excision of U and also oxidized pyrimidines such as 5-hydroxycytosine (5-OHC).…”