The alpha/beta fold uracil DNA glycosylases: a common origin with diverse fates

Abstract

Background: Uracil DNA glycosylases (UDGs) are major repair enzymes that protect DNA from mutational damage caused by uracil incorporated as a result of a polymerase error or deamination of cytosine. Four distinct families of UDGs have been identified, which show very limited sequence similarity to each other, although two of them have been shown to possess the same structural fold. The structural and evolutionary relationships between the rest of the UDGs remain uncertain.

Results: Using sequence profile searches, multiple alignment analysis and protein structure comparisons, we show here that all known UDGs possess the same fold and must have evolved from a common ancestor. Although all UDGs catalyze essentially the same reaction, significant changes in the configuration of the catalytic residues were detected within their common fold, which probably results in differences in the biochemistry of these enzymes. The extreme sequence divergence of the UDGs, which is unusual for enzymes with the same principal activity, is probably due to the major role of the uracil-flipping caused by the conformational strain enacted by the enzyme on uracil-containing DNA, as compared with the catalytic action of individual polar residues. We predict two previously undetected families of UDGs and delineate a hypothetical scenario for their evolution.

Conclusions: UDGs form a single protein superfamily with a distinct structural fold and a common evolutionary origin. Differences in the catalytic mechanism of the different families combined with the construction of the catalytic pocket have, however, resulted in extreme sequence divergence of these enzymes.

Figure 1

Multiple alignment of the UDG superfamily. The secondary-structure elements of the core UDG fold are shown in color above the multiple alignment. Some nonconserved elements in the MUG structure from E. coli are indicated in gray. The coloring of the alignment positions is according to the 85% consensus that includes the following categories of amino acid residues: h, hydrophobic, l, aliphatic, a, aromatic, shaded yellow (YFWLIVMA); s, small, individual letters colored green (SAGTVPNHD); p, polar, colored purple (STQNEDRKH); u, tiny, shaded green (GAS); and b, big, shaded gray (KREQWFYLMI). Af, Archaeoglobus fulgidus; Bb, Borrelia burgdorferi; Bs, Bacillus subtilis; Cj, Campylobacter jejuni; Ct, Chlamydia trachomatis; Dm, Drosophila melanogaster; Dr, Deinococcus radiodurans; Ec, Escherichia coli; Hi, Haemophilus influenzae; Hp, Helicobacter pylori; Hs, Homo sapiens; Mtu, Mycobacterium tuberculosis; Ph, Pyrococcus horikoshii; Rp, Rickettsia prowazekii; Sc, Saccharomyces cerevisiae; Scoel, Streptomyces coelicolor; Sp, Schizosaccharomyces pombe; Ssp, Synechocystis sp.; Tp, Treponema pallidum; Uu, Ureaplasma urealyticum; Yp, Yersinia pestis. The numbers at each end of each sequence are amino-acid positions and indicate the extent of the domain in each protein. The numbers within the alignment indicate inserts that have not been shown. The conserved motifs discussed in the text are designated I, II and III; the conserved aromatic (aliphatic) residue involved in the stacking interaction with uracil is indicated by an asterisk.

Figure 2

The topology of the UDG superfamily core fold, with the conserved and unique features of different families. The core secondary-structure elements of the UDG fold are colored as in Figure 1 and numbered according to their order in the sequence. The elements observed only in the MUGs are shown in gray. The conserved motif I occurs after strand 1 and motif II occurs after strand 4 and forms the active-site pocket in the three-dimensional structure.

Figure 3

A hypothetical evolutionary scenario for the UDG superfamily. The different families are shown in different colors and potential order and lineage of derivation is indicated on the standard phylogenetic model for the three domains of life. The representation of the active-site pocket residues typical of that set is shown next to each class at the point of derivation. The first position is the general base represented by an aspartate in the UNGs, the second position is the uracil/cytosine discrimination site occurring after the core strand 2, and the third position is typically represented by a histidine that acts as an electrophile. The X at a given position denotes lack of conservation. In some of the AUDGs and the DRUDGs, a glutamate could function as alternative general base.