Ribonucleotide reductases catalyze the formation of deoxyribonucleotides by the reduction of the corresponding ribonucleotides. Eukaryotic ribonucleotide reductases are ␣ 2 ␤ 2 tetramers; each of the larger, ␣ subunits possesses binding sites for substrate and allosteric effectors, and each of the smaller, ␤ subunits contains a binuclear iron complex. The iron complex interacts with a specific tyrosine residue to form a tyrosyl free radical which is essential for activity. Previous work has identified two genes in the yeast Saccharomyces cerevisiae, RNR1 and RNR3, that encode ␣ subunits and one gene, RNR2, that encodes a ␤ subunit. Here we report the identification of a second gene from this yeast, RNR4, that encodes a protein with significant similarity to the ␤-subunit proteins. The phenotype of rnr4 mutants is consistent with that expected for a defect in ribonucleotide reductase; rnr4 mutants are supersensitive to the ribonucleotide reductase inhibitor hydroxyurea and display an S-phase arrest at their restrictive temperature. rnr4 mutant extracts are deficient in ribonucleotide reductase activity, and this deficiency can be remedied by the addition of exogenous Rnr4p. As is the case for the other RNR genes, RNR4 is induced by agents that damage DNA. However, Rnr4p lacks a number of sequence elements thought to be essential for iron binding, and mutation of the critical tyrosine residue does not affect Rnr4p function. These results suggest that Rnr4p is catalytically inactive but, nonetheless, does play a role in the ribonucleotide reductase complex.Ribonucleotide reductases catalyze the formation of deoxyribonucleotides by the reduction of the corresponding ribonucleotides. Three classes of ribonucleotide reductases have been well characterized (24). Class I enzymes are found in all eukaryotes and some prokaryotes. The best-studied class I enzyme is the Escherichia coli ribonucleotide reductase (10, 30), an ␣ 2 ␤ 2 tetramer that can be decomposed to two catalytically inactive homodimers, R1 (␣ 2 ) and R2 (␤ 2 ). Each of the larger ␣ subunits possesses binding sites for substrate and allosteric effectors and also contains several redox-active cysteine residues. Each of the smaller ␤ subunits contains a binuclear Fe(III) complex. The X-ray structure of E. coli R2 reveals that the iron ions are bridged by both an O 2Ϫ ion and the carboxyl group of a glutamate residue (22). Each iron is further liganded by two carboxyl oxygen atoms from aspartate or glutamate residues, a histidine N␦ residue, and a water molecule. The recently solved structure of the mouse R2 protein indicates that the iron-binding center of eukaryotic proteins is similar to that of the E. coli protein (17). The iron complex interacts with a specific tyrosine residue to form a tyrosyl free radical which is essential for activity. The enzyme is inhibited by hydroxyurea, which specifically quenches the tyrosyl radical (19).Amino acid sequence alignments of the class 1 R2 proteins from different species identify 16 residues that are conserved in a...

Survivin is highly expressed in human gastric cancer cells and correlated with chemoresistance and poor prognosis. in this study, we explored the effect of adeno-associated virus (aaV)-mediated survivin dominant mutant Thr34ala (raaV-SurMut(T34a)) on gastric cancer growth. aaV-Sur-Mut (T34a) virus was generated and purified using the aaV vector paM/ Cag-WPRe.poly(a) vector. Cell proliferation was determined by the 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay. apoptosis was determined by Flow cytometry and Terminal deoxynucleotidyl transferase duTP nick end labeling. gene expression was determined by Western blot and immunohistochemistry. Tumor growth was evaluated using a xenograft mouse model. Overexpression of survivin promoted cell growth of gastric cancer cells. infection of raaVSur-Mut(T34a) virus inhibited cell proliferation, induced apoptosis and sensitized gastric cancer cells to 5-Fluorouracil in vitro. Treatment of raaV-Sur-Mut(T34a) significantly inhibited cell proliferation, induced apoptosis and inhibited gastric cancer growth in vivo. Our results suggest that the combination of raaV-Sur-Mut(T34a) and chemotherapy may be a new approach for gastric cancer therapy.