HUMAN DUTP PYROPHOSPHATASE - URACIL RECOGNITION BY A BETA-HAIRPIN ANDACTIVE-SITES FORMED BY 3 SEPARATE SUBUNITS

Background: The essential enzyme dUTP pyrophosphatase (dUTPase) is exq uisitely specific for dUTP and is critical for the fidelity of DNA rep lication and repair. dUTPase hydrolyzes dUTP to dUMP and pyrophosphate , simultaneously reducing dUTP levels and providing the dUMP for dTTP biosynthesis. A high cellular dTTP:dUTP ratio is essential to avoid ur acil incorporation into DNA, which would lead to strand breaks and cel l death. We report the first detailed atomic-resolution structure of a eukaryotic dUTPase, human dUTPase, and complexes with the uracil-cont aining deoxyribonucleotides, dUMP, dUDP and dUTP. Results: The crystal structure reveals that each subunit of the dUTPase trimer folds into an eight-stranded jelly-roll beta barrel, with the C-terminal beta str ands interchanged among the subunits. The structure is similar to that of the E. coli enzyme, despite low sequence homology between the two enzymes. The nucleotide complexes reveal a simple and elegant way for a beta hairpin to recognize specific nucleic acids: uracil is inserted into a distorted antiparallel beta hairpin and hydrogen bonds entirel y to main-chain atoms. This interaction mimics DNA base pairing, selec ting uracil over cytosine and sterically precluding thymine and ribose binding, Residues from the second subunit interact with the phosphate groups and a glycine-rich C-terminal tail of the third subunit caps t he substrate-bound active site, causing total complementary enclosure of substrate. To our knowledge, this is the first documented instance of all three subunits of a trimeric enzyme supplying residues that are critical to enzyme function and catalysis. Conclusions: The dUTPase n ucleotide-binding sites incorporate some features of other nucleotide- binding proteins and protein kinases, but seem distinct in sequence an d architecture, The novel nucleic acid base recognition motif appears ancient; higher order structures, such as the ribosome, may have evolv ed from a motif of this kind. These uracil-beta-hairpin interactions a re an obvious way for peptides to become early coenzymes in an RNA wor ld, providing a plausible link to the protein-DNA world. Within the be ta hairpin, there is a tyrosine corner motif that normally specifies b eta-arch connections; this tyrosine motif was apparently recruited to discriminate against ribonucleotides, more recently than the evolution of the beta hairpin itself.