Structural insights into the catalytic mechanism of phosphate ester hydrolysis by dUTPase.

dUTPase is essential to keep uracil out of DNA. Crystal structures of substrate (dUTP and alpha,beta-imino-dUTP) and product complexes of wild type and mutant dUTPases were determined to reveal how an enzyme responsible for DNA integrity functions. A kinetic analysis of wild type and mutant dUTPases was performed to obtain relevant mechanistic information in solution.

Developmental regulation of dUTPase in Drosophila melanogaster.

dUTPase prevents uracil incorporation into DNA by strict regulation of the cellular dUTP:dTTP ratio. Lack of the enzyme initiates thymineless cell death, prompting studies on enzyme regulation. We investigated expression pattern and localization of Drosophila dUTPase.

Altered active site flexibility and a structural metal-binding site in eukaryotic dUTPase: kinetic characterization, folding, and crystallographic studies of the homotrimeric Drosophila enzyme.

dUTPase is responsible for preventive DNA repair via exclusion of uracil. Developmental regulation of the Drosophila enzyme is suggested to be involved in thymine-less apoptosis. Here we show that in addition to conserved dUTPase sequence motifs, the gene of Drosophila enzyme codes for a unique Ala-Pro-rich segment.

dUTPase and nucleocapsid polypeptides of the Mason-Pfizer monkey virus form a fusion protein in the virion with homotrimeric organization and low catalytic efficiency.

Betaretroviruses encode dUTPase, an essential factor in DNA metabolism and repair, in the pro open reading frame located between gag and pol. Ribosomal frame-shifts during expression of retroviral proteins provide a unique possibility for covalent joining of nucleocapsid (NC) and dUTPase within Gag-Pro polyproteins. By developing an antibody against the prototype betaretrovirus Mason-Pfizer monkey virus dUTPase, we demonstrate that i) the NC-dUTPase fusion protein exists both within the virions and infected cells providing the only form of dUTPase, and ii) the retroviral protease does not cleave NC-dUTPase either in the virion or in vitro.