Keeping uracil out of DNA: physiological role, structure and catalytic mechanism of dUTPases

Abstract

The thymine-uracil exchange constitutes one of the major chemical differences between DNA and RNA. Although these two bases form the same Watson-Crick base pairs with adenine and are equivalent for both information storage and transmission, uracil incorporation in DNA is usually a mistake that needs to be excised. There are two ways for uracil to appear in DNA: thymine replacement and cytosine deamination. Most DNA polymerases readily incorporate dUMP as well as dTMP depending solely on the availability of the d(U/T)TP building block nucleotides. Cytosine deamination results in mutagenic U:G mismatches that must be excised. The repair system, however, also excises U from U:A "normal" pairs. It is therefore crucial to limit thymine-replacing uracils.dUTP is constantly produced in the pyrimidine biosynthesis network. To prevent uracil incorporation into DNA, representatives of the dUTP nucleotidohydrolase (dUTPase) enzyme family eliminate excess dUTP. This Account describes recent studies that have provided important detailed insights into the structure and function of these essential enzymes.dUTPases typically possess exquisite specificity and display an intriguing homotrimer active site architecture. Conserved residues from all three monomers contribute to each of the three active sites within the dUTPase. Although even dUTPases from evolutionarily distant species possess similar structural and functional traits, in a few cases, a monomer dUTPase mimics the trimer structure through an unusual folding pattern. Catalysis proceeds by way of an SN2 mechanism; a water molecule initiates in-line nucleophilic attack. The dUTPase binding pocket is highly specific for uracil. Phosphate chain coordination involves Mg2+ and is analogous to that of DNA polymerases. Because of conformational changes in the enzyme during catalysis, most crystal structures have not resolved the residues in the C-terminus. However, recent high-resolution structures are beginning to provide in-depth structural information about this region of the protein.The dUTPase family of enzymes also shows promise as novel targets for anticancer and antimicrobial therapies. dUTPase is upregulated in human tumor cells. In addition, dUTPase inhibitors could also fight infectious diseases such as malaria and tuberculosis. In these respective pathogens, Plasmodium falciparum and Mycobacterium tuberculosis, the biosynthesis of dTMP relies exclusively on dUTPase activity.

FIGURE 1

(A) Watson-Crick base pairing between adenine and thymine (i.e., 5-methyl-uracil) and (B) de novo biosynthesis of dTMP.

FIGURE 2

Uracil-excision repair. The deaminated cytosine is excised by uracil-DNA glycosylase. AP endonuclease nicks the DNA phosphodiester backbone at the abasic site, creating a free 3′-OH. 5′-phosphodiesterase removes the sugar from the abasic site, and the gap is filled by DNA polymerase. Ligase completes the repair.

FIGURE 3

A. Dual role of dUTPase. In the absence of dUTPase (i.e., when dUTP/dTTP ratio is high), thymine-replacing uracils will be reincorporated during base excision repair (BER), but cytosine deamination can be correctly repaired (underlined bases U, C). B. De novo and salvage pathways for dTTP biosynthesis. Enzymes not present in Mycobacteria and Plasmodia are crossed out.

FIGURE 4

Sequence alignments of dUTPases. Conserved motifs appear in white lettering on black background. Arrows indicate conserved residues shown in Figure 5–Figure 7.

FIGURE 5

Active site close-up in M. tuberculosis dUTPase (2PY4). Substrate coordinating residues are labeled. Roman numerals stand for respective conserved motifs. Dashed lines indicate H-bonds; shaded rectangles indicate aromatic overlaps. In this dUTPase, the aromatic phenylalanine within motif 5 (see Figure 4) is replaced with a histidine.

FIGURE 6

Mechanism of dUTPase-catalyzed dUTP hydrolysis. (A) Reaction scheme of dUTP hydrolysis showing Mg(II) coordination to the triphosphate chain of the substrate. Mg(II) probably dissociates from the enzyme with the product PPi and not with dUMP because no Mg(II) could be observed in dUTPase–dUMP crystal structures. (B) Active site of human dUTPase (2HQU). Conserved residues responsible for coordination of the relevant water molecules and the phosphate chain are shown as sticks connected to the ribbon model of the entire protein. Non-carbon atoms are color-coded as oxygen, red; nitrogen, blue; phosphorus, orange; and Mg, light green. Residues of monomers B and C are shown in pink and green, respectively. Substrate carbons are yellow. H-bonds are shown as dashed lines. The catalytic water molecule (W_cat) is positioned for an in-line attack on the αP.

FIGURE 7

Catalytically competent and noncompetent conformation of the bound substrate. (A) dUDP (teal) and α,β-imido-dUTP (dUPNPP, magenta) complexed human dUTPase crystal structures (PDB IDs 1 Q5H and 2HQU) are superimposed to show relevant differences between the catalytically noncompetent trans (dUDP) and competent gauche (dUPNPP) binding modes within the active site. Surface and ribbon models of the protein are subunit-color-coded (A, yellow; B, green; C, blue). (B) Slab view of panel A to reveal the available inner hole that provides ample conformational space for the phosphate chain.