G 1 ͞S and G 2 ͞M cell cycle checkpoints maintain genomic stability in eukaryotes in response to genotoxic stress. We report here both genetic and functional evidence of a Gadd45-mediated G 2 ͞M checkpoint in human and murine cells. Increased expression of Gadd45 via microinjection of an expression vector into primary human fibroblasts arrests the cells at the G 2 ͞M boundary with a phenotype of MPM2 immunopositivity, 4n DNA content and, in 15% of the cells, centrosome separation. The Gadd45-mediated G 2 ͞M arrest depends on wild-type p53, because no arrest was observed either in p53-null Li-Fraumeni fibroblasts or in normal fibroblasts coexpressed with p53 mutants. Increased expression of cyclin B1 and Cdc25C inhibited the Gadd45-mediated G 2 ͞M arrest in human fibroblasts, indicating that the mechanism of Gadd45-mediated G 2 ͞M checkpoint is at least in part through modulation of the activity of the G 2 -specific kinase, cyclin B1͞p34 cdc2 . Genetic and physiological evidence of a Gadd45-mediated G 2 ͞M checkpoint was obtained by using GADD45-deficient human or murine cells. Human cells with endogenous Gadd45 expression reduced by antisense GADD45 expression have an impaired G 2 ͞M checkpoint after exposure to either ultraviolet radiation or methyl methanesulfonate but are still able to undergo G 2 arrest after ionizing radiation. Lymphocytes from gadd45-knockout mice (gadd45 ؊͞؊) also retained a G 2 ͞M checkpoint initiated by ionizing radiation and failed to arrest at G 2 ͞M after exposure to ultraviolet radiation. Therefore, the mammalian genome is protected by a multiplicity of G 2 ͞M checkpoints in response to specific types of DNA damage.Mammalian cells have evolved an intricate defense network to maintain genomic integrity by preventing the fixation of permanent damage from endogenous and exogenous mutagens. Cellcycle checkpoints, a major genomic surveillance mechanism, exist at the G 1 ͞S and G 2 ͞M transitions that are regulated in response to DNA damage (1). Defects in these steps may result in a mutator phenotype that is associated with tumorigenesis.Tumor suppressor gene product p53 is implicated to be one of the essential components of cell-cycle checkpoints (2-5). p53 is a transcription factor that up-regulates a number of important cell cycle-modulating genes, including p21 WAF1͞CIP1͞SDI1

Fe2؉ is now shown to weaken binding between ferritin and mitochondrial aconitase messenger RNA noncoding regulatory structures ((iron-responsive element) (IRE)-RNAs) and the regulatory proteins (IRPs), which adds a direct role of iron to regulation that can complement the well known regulatory protein modification and degradative pathways related to iron-induced mRNA translation. We observe that the Iron (e.g. ferrous sulfate, ferric citrate, and hemin) added to animal cells changes translation rates of messenger RNAs encoding proteins of iron traffic and oxidative metabolism (1-4). To cross cell membranes, iron ions are transported by membrane proteins such as DMT1 or carried on proteins such as transferrin. Inside the cells, iron is mainly in heme, FeS clusters, non-heme iron cofactors of proteins, and iron oxide minerals coated by protein nanocages (ferritins). Iron in transit is thought to be Fe 2ϩ in labile "pools" accessible to small molecular weight chelators, and/or bound loosely by chaperones.When iron concentrations in the cells increase, a group of mRNAs with three-dimensional, noncoding structures in the 5Ј-untranslated region (UTR) 3 are derepressed (Fig. 1A), i.e. the fraction of the mRNAs in mRNA⅐repressor protein complexes, which inhibit ribosome binding, decreases and the fraction of the mRNAs in polyribosomes increases (5-7). The three-dimensional, noncoding mRNA structure, representing a family of related structures, is called the iron-responsive element, or IRE, and the repressors are called iron regulatory proteins (IRPs). Together they are one of the most extensively studied eukaryotic messenger RNA regulatory systems (1-4). In addition to large numbers of cell studies, structures of IRE-RNAs are known from solution NMR (8 -12), and the RNA⅐protein complex from x-ray crystallography (13). Recent data indicate that demetallation of IRP1 and disruption of the [4Fe-4S] cluster that inhibits IRP1 binding to RNA will be enhanced by phosphorylation and low iron concentrations (1, 2, 14 -16). Such results can explain the increased IRP1 binding to IRE-mRNAs and increased translational repression when iron concentrations are abnormally low. However, the mechanism to explain dissociation of IRE-RNA⅐IRP complexes, thereby allowing ribosome assembly and increased proteosomal degradation of IRPs (1, 2, 14, 15) (Fig. 1A), when high iron concentrations are abnormally high, is currently unknown.Metal ion binding changes conformation and function of most RNA classes, e.g. rRNA (17), tRNA (18, 19), ribozymes (20 -23), riboswitches (24, 25), possibly hammerhead mRNAs in mammals (26), and proteins. Although the effects of metal ion binding on eukaryotic mRNAs have not been extensively studied, Mg 2ϩ is known to cause changes in conformation, shown by changes in radical cleavage sites of IRE-RNA with 1,10-phenanthrolene-iron and proton shifts in the one-dimensional NMR spectrum (12, 27). The Mg 2ϩ effects are observed at low magnesium concentrations (0.1-0.5 mM) and low molar stoichiometries (1:1 and 2:1 ϭ Mg:...

Iron increases synthesis rates of proteins encoded in iron-responsive element (IRE)-mRNAs; metabolic iron ("free," "labile") is Fe 2þ . The noncoding IRE-RNA structure, approximately 30 nt, folds into a stem loop to control synthesis of proteins in iron trafficking, cell cycling, and nervous system function. IRE-RNA riboregulators bind specifically to iron-regulatory proteins (IRP) proteins, inhibiting ribosome binding. Deletion of the IRE-RNA from an mRNA decreases both IRP binding and IRP-independent protein synthesis, indicating effects of other "factors." Current models of IRE-mRNA regulation, emphasizing iron-dependent degradation/modification of IRP, lack answers about how iron increases IRE-RNA/IRP protein dissociation or how IRE-RNA, after IRP dissociation, influences protein synthesis rates. However, we observed Fe 2þ (anaerobic) or Mn 2þ selectively increase the IRE-RNA/IRP K D . Here we show: (i) Fe 2þ binds to the IRE-RNA, altering its conformation (by 2-aminopurine fluorescence and ethidium bromide displacement); (ii) metal ions increase translation of IRE-mRNA in vitro; (iii) eukaryotic initiation factor (eIF)4F binds specifically with high affinity to IRE-RNA; (iv) Fe 2þ increased eIF4F/IRE-RNA binding, which outcompetes IRP binding; (v) exogenous eIF4F rescued metal-dependent IRE-RNA translation in eIF4F-depeleted extracts. The regulation by metabolic iron binding to IRE-RNA to decrease inhibitor protein (IRP) binding and increase activator protein (eIF4F) binding identifies IRE-RNA as a riboregulator.ferrous ion regulation | metabolic riboregulator I ron increases rates of ferritin protein synthesis in animals by facilitating messenger RNA/ribosome binding; metabolic iron (i.e., "labile" or "free" iron in cells) is considered to be ferrous (1). The iron response requires a noncoding riboregulator called the "iron-responsive element" (IRE), which is approximately 30 nt, folded into a distorted, bulged helix loop (2-5). This riboregulatory structure is also found in mRNAs for proteins of iron traffic (6-9), cell cycling (10), and the nervous system (11). IRP proteins bind with different stabilities to IRE-RNAs of the IRE-RNA family (12, 13), creating a graded or hierarchal set of mRNA responses to iron in vivo. Deletion of the 30 nt IRE-RNA not only removes IRP regulation but also decreases the rate of IRP-independent protein synthesis (14). A number of current models of IRE-RNA/IRP regulation feature iron-dependent degradation/modification of the IRP proteins as the main control point (8,9,15,16). Such models do not answer two important questions: (i) How does iron increase release of IRP protein for [4Fe-4S]-modification and/or degradation? Overlap of the IRE-RNA and the Fe-S binding sites on IRP1 prevents Fe-S insertion in the IRP1/IRE-RNA complex (5). (ii) How does the IRE-RNA control rates of IRP-independent protein synthesis (14)? In an earlier study, we showed that Fe 2þ ions (anaerobic) selectively increased the dissociation constant for the IRE-RNA/IRP1 complex in solution (12). Here we r...