Recently, a homologue of the small subunit of mammalian ribonucleotide reductase (RNR) was discovered, called p53R2. Unlike the well characterized S phasespecific RNR R2 protein, the new form was induced in response to DNA damage by the p53 protein. Because the R2 protein is specifically degraded in late mitosis and absent in G o /G 1 cells, the induction of the p53R2 protein may explain how resting cells can obtain deoxyribonucleotides for DNA repair. However, no direct demonstration of RNR activity of the p53R2 protein was presented and furthermore, no corresponding RNR large subunit was identified. In this study we show that recombinant, highly purified human and mouse p53R2 proteins contain an iron-tyrosyl free radical center, and both proteins form an active RNR complex with the human and mouse R1 proteins. UV irradiation of serumstarved, G o /G 1 -enriched mouse fibroblasts, stably transformed with an R1 promoter-luciferase reporter gene construct, caused a 3-fold increase in luciferase activity 24 h after irradiation, paralleled by an increase in the levels of R1 protein. Taken together, our data indicate that the R1 protein can function as the normal partner of the p53R2 protein and that an R1-p53R2 complex can supply resting cells with deoxyribonucleotides for DNA repair.Ribonucleotide reductase (RNR) 1 catalyzes de novo synthesis of deoxyribonucleotides from the corresponding ribonucleotides (1). This is the rate-limiting step in DNA precursor synthesis, and it is regulated at many different levels in the mammalian cell: (i) by allosteric control of the activity and specificity of ribonucleotide reductase by nucleoside triphosphate effectors (2), (ii) by the S phase-dependent transcription of the ribonucleotide reductase genes (3), and (iii) by the rapid, proteasomedependent proteolysis of the small subunit in late mitosis (4).Errors in the allosteric control lead to unbalanced dNTP pools, misincorporation of deoxyribonucleotides in DNA, genetic abnormalities or cell death (5). The mammalian ribonucleotide reductase consists of a 1:1 complex of two homodimeric nonidentical subunits called proteins R1 and R2. The 90-kDa R1 protein contains the catalytic site, the binding sites for the allosteric effectors, and redox active disulfides that participate in the reduction of the substrates, while the 45-kDa R2 protein contains an iron center-generated tyrosyl free radical essential for catalysis (6). Transcription of both the R1 and R2 genes is S-phase specific but only the R2 protein shows S-phase-specific expression (3, 7). The levels of the R1 protein are in excess and almost constant during the cell cycle in proliferating cells because of a long half-life of the protein (Ͼ24 h) (8, 9). Therefore, the cell cycle-dependent activity of ribonucleotide reductase is controlled by the synthesis and degradation of the R2 protein. This synthesis starts in early S phase, and then the R2 protein

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The pore-forming domains of members of a family of bacterial toxins, colicins N and A, share > 50% sequence identity, identical folds and yet display strikingly different behavior in acid conditions. At low pH colicin A forms a molten globule state while colicin N retains a native fold. This is relevant to in vivo activity since colicin A requires acidic phospholipids for its toxic activity but colicin N does not. The pI of colicin A (5.25) is far lower than that of colicin N (10.2) because colicin A contains seven extra aspartate residues. We first introduced separately each of these acidic amino acids into homologous sites in colicin N, but none caused destabilization at low pH. However, in the reverse experiment, the sequential replacement of these acidic side chains of colicin A by alanine revealed six sites where this change destabilized the protein at neutral pH. Some of these residues, which each contribute less than 4% to the total negative charge, appear to stabilize the protein via a network of hydrogen bonds and charge pairs which are sensitive to protonation. Other residues have no clear interactions that explain their importance. The colicin A is thus a protein that relies upon acid sensitive interactions for its stability at neutral pH and its in vivo activity.

Colicin N kills sensitive Escherichia coli cells by first binding to its trimeric receptor (OmpF) via its receptor binding domain. It then uses OmpF to translocate across the outer membrane and in the process it also needs domains II and III of the protein TolA. Recent studies have demonstrated sodium dodecyl sulfate- (SDS) dependent complex formation between trimeric porins and TolA-II. Here we demonstrate that colicin N forms similar complexes with the same trimeric porins and that this association is unexpectedly solely dependent upon the pore-forming domain (P-domain). No binding was seen with the monomeric porin OmpA. In mixtures of P-domain and TolA with OmpF porin, only binary and no ternary complexes were observed, suggesting that binding of these proteins to the porin is mutually exclusive. Pull-down assays in solution show that porin-P-domain complexes also form in the presence of outer membrane lipopolysaccharide. This indicates that an additional colicin-porin interaction may occur within the outer membrane, one that involves the colicin pore domain rather than the receptor-binding domain. This may help to explain the role of porins and TolA-II in the later stages of colicin translocation.