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Uracil in DNA: Prevention, Recognition, Repair, and Signal Transduction
Summary: Using integrated tools of biochemistry, structural biology, and cell biology, Beáta Vértessy investigates DNA repair and signaling pathways related to spontaneous DNA damage signals.
A major paradox of life is that DNA, though a macromolecule of inherent significant chemical reactivity, is responsible for the stable and faithful storage of genetic information. Physiological conditions, such as normal levels of activated oxygen species, water, and several additional reactive metabolites, induce chemical modifications within DNA with high frequency. These so-called spontaneous damage reactions lead to mutagenic changes within base and sugar moieties and may also lead to cleavage of the DNA backbone. Cytosine deamination, one of the most frequent spontaneous DNA damage reactions, leads to the appearance of uracil in DNA. This reaction changes the readout of genetic information because uracil is a thymine analogue that, if left unrepaired, will form a Watson-Crick base pair with adenine during the next replication cycle. The enzyme that catalyzes uracil-excision repair is uracil-DNA glycosylase (UDG).
Thymine-replacing uracils can also be easily incorporated by DNA polymerases if the cellular ratio of the concentration of dTTP to dUTP (the building block nucleotides) is too low. It is thus essential to finely regulate dUTP levels; this is the physiological role of the enzyme family of dUTPases. Therefore, the key enzymes that control the two pathways that lead to the appearance of uracil in DNA (cytosine deamination and thymine replacement) are dUTPases—a preventive factor—and UDGs—the cure.
The dUTPase Family
Fulfilling a dual role, dUTPases catalyze the cleavage of dUTP into dUMP and inorganic pyrophosphate. The enzyme efficiently reduces the cellular level of dUTP; strict specificity for cleavage of the phosphate chain at the α-β linkage renders the reaction energetically highly favorable. The product of dUTPase-catalyzed cleavage is dUMP, the precursor for dTMP biosynthesis. The dUTPases are ubiquitous—they are encoded in all free-living organisms and in several DNA viruses (herpesviruses) and retroviruses (lentiviruses, betaretroviruses).
Deficiency or lack of dUTPase has major consequences. The increased dUTP/dTTP ratio results in a high level of dUMP incorporation into DNA. Uracil-substituted DNA is subjected to uracil-excision repair. However, thymine-replacing uracils cannot be successfully removed if the dUTP/dTTP ratio is high because the uracils are reincorporated during repair synthesis. Transformation of uracil-excision repair into a hyperactive but futile cycle finally leads to cell death via double-stranded DNA breaks (thymine-less cell death). Accordingly, dUTPase knockouts are lethal in both Escherichia coli and yeast. In human cells, siRNA-mediated partial silencing of dUTPase results in increased sensitivity to 5′-fluorodeoxyuridine, a chemotherapeutic agent frequently used in anticancer therapies that also perturbs dUTP/dTTP levels by inhibiting thymidylate synthase. This observation indicates that thymidylate metabolism may be affected at different levels and suggests that combination therapies directed against more than one enzyme target in this pathway may have synergistic effects.
We determined the crystal structures of substrate (dUTP and α,β-imino-dUTP) and product complexes of wild-type and mutant dUTPases to reveal how this enzyme, which is responsible for DNA integrity, functions. To obtain relevant mechanistic information in solution, we performed a kinetic analysis of wild-type and mutant dUTPases. We showed that substrate hydrolysis is initiated via an in-line nucleophile attack of a water molecule oriented by an activating conserved aspartate residue. We showed that substrate binding in a catalytically competent conformation is achieved by (1) multiple interactions with catalysis-assisting Mg2+; (2) concerted motion, which does not occur in the apoenzyme, of residues from three conserved enzyme motifs; and (3) an intricate hydrogen-bonding network that includes several water molecules in the active site. These results provided an understanding of the catalytic role of conserved residues in dUTPases.
In Mason-Pfizer monkey cells infected with betaretrovirus, the homotrimeric fusion protein nucleocapsid-dUTPase combines domains that participate in RNA/DNA folding, reverse transcription, and DNA repair. The structural organization of the fusion protein remained obscured by the N- and C-terminal flexible segments of dUTPase and the linker region connecting these two domains, which are invisible in electron density maps.
Small-angle X-ray scattering revealed that, upon oligonucleotide binding, the nucleocapsid (NC) domains adopt the trimeric symmetry of dUTPase. High-resolution X-ray structures together with molecular modeling indicated that fusion with NC domains dramatically alters the conformation of the flexible C terminus by perturbing the orientation of a critical β-strand. We showed that, as a consequence, the C-terminal segment is capable of double-backing on the active site of its own monomer and is stabilized by noncovalent interactions formed with the N-terminal segment. We also showed that this cofolding of the dUTPase terminal segments, not observable in other homologous enzymes, was attributable to the presence of the fused NC domain. Betaretroviruses have found a unique economic solution by encoding a shortened, though effective, dUTPase fused to the NC protein in their very limited genome. The fusion protein may thus efficiently decrease dUTP pools exactly where reverse transcription occurs and thus avoid wasting time and energy by hydrolyzing all cellular dUTP.
Uracil in DNA: A Possible Signal During Metamorphosis
We investigated the expression pattern and localization of Drosophila dUTPase. Similar to the human enzyme, two isoforms of the fly enzyme were identified at the mRNA and protein levels. During larval stages, we found a drastic decline in dUTPase expression at the protein level, whereas dUTPase mRNAs display a constitutive character throughout development. We identified a putative nuclear localization signal in one of the two isoforms. However, immunohistochemistry of ovaries and embryos did not show a clear correlation between the presence of this signal and subcellular localization of the protein, suggesting that the latter may be perturbed by additional factors. Results were in agreement with a multilevel regulation of dUTPase in the Drosophila proteome, possibly involving several interacting protein partners of the enzyme. Using independent approaches, we verified the existence of such macromolecular partners.
We found that the absence of dUTPase in Drosophila larvae occurs in parallel with the absence of the main UDG isoform from the Drosophila genome. Fruitfly larvae therefore lack both dUTPase and UDG; under these conditions, larval tissues may accumulate dUMP-substituted DNA. We investigated whether larval tissues contain proteins that specifically recognize dUMP-substituted DNA (uracil-DNA). We showed that the most abundant hit of pull-down experiments on uracil-DNA is the protein product of the Drosophila melanogaster gene CG18410. This protein binds to both uracil-DNA and normal DNA but degrades only uracil-DNA. The novel protein factor, termed uracil-DNA degrading factor (UDE), has no detectable homology to any proteins except a group of sequences present in genomes of pupating insects. Comparison of UDE-like sequences from pupating insects identified four conserved motifs, the first of which is duplicated. Limited proteolysis experiments indicated that DNA binding may occur along the conserved motifs and induce protein conformational transition(s), resulting in significant protection against multiple proteases. During fruitfly development, UDE protein is undetectable in the embryo, most of the larval stages, and the imago, whereas it is strongly upregulated immediately before pupation and shows its highest level at the prepupal/pupal transition. In Schneider 2 cells, the UDE mRNA transcript is upregulated by treatment with ecdysone. UDE protein localizes to the nucleus of Drosophila cells. With its novel sequence, unique specificity, cellular distribution, and developmental control, UDE represents a new class of proteins that recognize and process dUMP-substituted DNA.
Last updated May 2007
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