Escherichia coli Orf135 protein is thought to be an enzyme that efficiently hydrolyzes oxidatively damaged nucleotides such as 2-hydroxy-dATP, 8-hydroxy-dGTP and 5-hydroxy-CTP, in addition to 5-methyl-dCTP, dCTP and CTP, thus preventing mutations in cells caused by unfavorable base pairing. Nucleotide pool sanitization by Orf135 is important since organisms are continually subjected to potential damage by reactive oxygen species produced during respiration. It is known that the frequency of spontaneous and H(2)O(2)-induced mutations is two to threefold higher in the orf135(-) strain compared with the wild-type. Orf135 is a member of the Nudix family of proteins which hydrolyze nucleoside diphosphate derivatives. Nudix hydrolases are characterized by the presence of a conserved motif, although they recognize various substrates and possess a variety of substrate binding pockets. We are interested in delineating the mechanism by which Orf135 recognizes oxidatively damaged nucleotides. To this end, we are investigating the tertiary structure of Orf135 and its interaction with substrate using NMR. Herein, we report on the (1)H, (13)C and (15)N resonance assignments of Orf135, which should contribute towards a structural understanding of Orf135 and its interaction with substrate.