Rnr1's role in telomere elongation cannot be replaced by Rnr3: a role beyond dNTPs?

Telomeres, the nucleoprotein complexes at the end of eukaryotic chromosomes, protect them from degradation and ensure the replicative capacity of cells. In most human tumors and in budding yeast, telomere length is maintained by the activity of telomerase, an enzyme that adds dNTPs according to an internal RNA template. The dNTPs are generated with the help of the ribonucleotide reductase (RNR) complex. We have recently generated strains lacking the large subunit of RNR, Rnr1, which were kept viable by the expression of RNR complexes containing the Rnr1 homolog, Rnr3. Interestingly, we found that these Rnr1-deficient strains have short telomeres that are stably maintained, but cannot become efficiently elongated by telomerase. Thus, a basic maintenance of short telomeres is possible under conditions, where Rnr1 activity is absent, but a sustained elongation of short telomeres fully depends on Rnr1 activity. We show that Rnr3 cannot compensate for this telomeric function of Rnr1 even when overall cellular dNTP values are restored. This suggests that Rnr1 plays a role in telomere elongation beyond increasing cellular dNTP levels. Furthermore, our data indicate that telomerase may act in two different modes, one that is capable of coping with the "end-replication problem" and is functional even in the absence of Rnr1 and another required for the sustained elongation of short telomeres, which fully depends on the presence of Rnr1. Supply of dNTPs for telomere elongation is provided by the Mec1ATR checkpoint, both during regular DNA replication and upon replication fork stalling. We discuss the implications of these results on telomere maintenance in yeast and cancer cells.

Ribonucleotides incorporated by the yeast mitochondrial DNA polymerase are not repaired.

Incorporation of ribonucleotides into DNA during genome replication is a significant source of genomic instability. The frequency of ribonucleotides in DNA is determined by deoxyribonucleoside triphosphate/ribonucleoside triphosphate (dNTP/rNTP) ratios, by the ability of DNA polymerases to discriminate against ribonucleotides, and by the capacity of repair mechanisms to remove incorporated ribonucleotides. To simultaneously compare how the nuclear and mitochondrial genomes incorporate and remove ribonucleotides, we challenged these processes by changing the balance of cellular dNTPs. Using a collection of yeast strains with altered dNTP pools, we discovered an inverse relationship between the concentration of individual dNTPs and the amount of the corresponding ribonucleotides incorporated in mitochondrial DNA, while in nuclear DNA the ribonucleotide pattern was only altered in the absence of ribonucleotide excision repair. Our analysis uncovers major differences in ribonucleotide repair between the two genomes and provides concrete evidence that yeast mitochondria lack mechanisms for removal of ribonucleotides incorporated by the mtDNA polymerase. Furthermore, as cytosolic dNTP pool imbalances were transmitted equally well into the nucleus and the mitochondria, our results support a view of the cytosolic and mitochondrial dNTP pools in frequent exchange.

Preventive DNA repair by sanitizing the cellular (deoxy)nucleoside triphosphate pool.

The occurrence of modified bases in DNA is attributed to some major factors: incorporation of altered nucleotide building blocks and chemical reactions or radiation effects on bases within the DNA structure. Several enzyme families are involved in preventing the incorporation of noncanonical bases playing a 'sanitizing' role. The catalytic mechanism of action of these enzymes has been revealed for a number of representatives in clear structural and kinetic detail. In this review, we focus in detail on those examples where clear evidence has been produced using high-resolution structural studies. Comparing the protein fold and architecture of the enzyme active sites, two main classes of sanitizing deoxyribonucleoside triphosphate pyrophosphatases can be assigned that are distinguished by the site of nucleophilic attack. In enzymes associated with attack at the α-phosphorus, it is shown that coordination of the γ-phosphate group is also ensured by multiple interactions. By contrast, enzymes catalyzing attack at the ß-phosphorus atom mainly coordinate the α- and the ß-phosphate only. Characteristic differences are also observed with respect to the role of the metal ion cofactor (Mg(2+) ) and the coordination of nucleophilic water. Using different catalytic mechanisms embedded in different protein folds, these enzymes present a clear example of convergent evolution.

Nucleic acid agonists for Toll-like receptor 7 are defined by the presence of uridine ribonucleotides.

Toll-like receptor 7 (TLR7) mediates innate responses by responding to viral RNA in endocytic compartments. However, the molecular pattern recognised by TLR7 and whether it differs between RNA of viral and self origin remains unclear. Here, we identify nucleic acids that act as TLR7 agonists for mouse and human cells. We show that uridine and ribose, the two defining features of RNA, are both necessary and sufficient for TLR7 stimulation, and that short single-stranded RNA (ssRNA) act as TLR7 agonists in a sequence-independent manner as long as they contain several uridines in close proximity. Consistent with the notion that TLR7 lacks specificity for sequence motifs, we show that it is triggered equally efficiently by viral or self RNA delivered to endosomes. Our results support the notion that TLR7 recognises uracil repeats in RNA and that it discriminates between viral and self ligands on the basis of endosomal accessibility rather than sequence.

Role of dNTPs in mutagenesis.

The induced mutation frequency by alkylating mutagen glycidyl methacrylate (GMA) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was investigated with or without perturbation of deoxyribonucleoside triphosphate (dNTP) pools; the influence of short treatment at different concentrations of GMA or MNNG on dNTP pools was also explored. The results indicated that the induced mutation frequency increased greatly at high dosages of mutagen (GMA approximately 64 micrograms/ml, MNNG approximately 8 micrograms/ml) and the perturbation on dNTP pools was carried out before the treatment of mutagen; the short treatment with mutagen could induce distinct fluctuations of dNTP pools, but different mutagen might have different effects on dNTP pools. According to the results of the present study and other reports in literature, we conclude that dNTP pools may be the targets of alkylating mutagens and the fluctuations of dNTP pools are closely associated with mutagenesis.

Role of deoxyribonucleoside triphosphates (dNTPs) in cell transformation.

Deoxyribonucleoside triphosphate (dNTP) pools were measured in normal BALB/c3T3 cells, transformation-treated cells and transformed cells with reverse-phase HPLC. The fluctuation of dNTP pools was similar after transformation treatment with alkylating mutagen glycidyl methacrylate (GMA) or Nmethyl-N'-nitro-N-nitrosoguanidine (MNNG). However, the gap between deoxyguanosine triphosphate + deoxyadenosine triphosphate (dGTP + dATP) pools and deoxythymidine triphosphate + deoxycytidine triphosphate (dTTP + dCTP) pools was greatly intensified. The measurements also indicated that the dNTP pools in transformed cells were quite different from those in normal cells. The results suggested that dNTP pools may play an important role in cell transformation.

dNTP pools imbalance as a signal to initiate apoptosis.

Fidelity in DNA synthesis and repair is largely dependent on a balanced supply of deoxynucleotide triphosphate (dNTP) pools. Results from different groups have shown that alterations in dNTP supply result in DNA fragmentation and cell death with characteristics of apoptosis. We have recently shown that in apoptosis driven by deprivation of interleukin-3 (IL-3) in a murine hemopoietic cell line, there is a rapid imbalance in the availability of dNTP that precedes DNA fragmentation. In these cells, dNTP pool balance is closely coupled to the function of the salvage pathway of dNTP synthesis. Apoptosis, induced by treatment of these cells with drugs that inhibit the de novo dNTP synthesis, is prevented when dNTP precursors are supplied through the salvage pathway. IL-3 regulates thymidine kinase activity, suggesting that alterations in dNTP metabolism after IL-3 deprivation could be a relevant event in the commitment of hemopoietic cells to apoptosis.

Protein import into mitochondria: the requirement for external ATP is precursor-specific whereas intramitochondrial ATP is universally needed for translocation into the matrix.

ATP is needed for the import of precursor proteins into mitochondria. However, the role of ATP and its site of action have been unclear. We have now investigated the ATP requirements for protein import into the mitochondrial matrix. These experiments employed an in vitro system that allowed ATP levels to be manipulated both inside and outside the mitochondrial inner membrane. Our results indicate that there are two distinct ATP requirements for mitochondrial protein import. ATP in the matrix is always needed for complete import of precursor proteins into this compartment, even when the precursors are presented to mitochondria in an unfolded conformation. In contrast, the requirement for external ATP is precursor-specific; depletion of external ATP strongly inhibits import of some precursors but has little or no effect with other precursors. A requirement for external ATP can often be overcome by denaturing the precursor with urea. We suggest that external ATP promotes the release of precursors from cytosolic chaperones, whereas matrix ATP drives protein translocation across the inner membrane.

Gap formation is associated with methyl-directed mismatch correction under conditions of restricted DNA synthesis.

A covalently closed, circular heteroduplex containing a G-T mismatch and a single hemimethylated d(GATC) site is subject to efficient methyl-directed mismatch correction in Escherichia coli extracts when repair DNA synthesis is severely restricted by limiting the concentration of exogenously supplied deoxyribonucleoside-5'-triphosphates or by supplementing reactions with chain-terminating 2',3'-dideoxynucleoside triphosphates. However, repair under these conditions results in formation of a single-strand gap in the region of the molecule containing the mismatch and the d(GATC) site. These findings indicate that repair DNA synthesis required for methyl-directed correction can initiate in the vicinity of the mispair, and they are most consistent with a repair reaction involving 3'----5' excision (or strand displacement) from the d(GATC) site followed by 5'----3' repair DNA synthesis initiating in the vicinity of the mismatch.

Factors determining the activity of 2',3'-dideoxynucleosides in suppressing human immunodeficiency virus in vitro.

Mitsuya and Broder [Proc. Natl. Acad. Sci. USA 83:1911-1915 (1986)] demonstrated that every purine (adenosine, guanosine, and inosine) and pyrimidine (cytidine and thymidine) nucleoside containing the 2',3'-dideoxyribose configuration, when evaluated against human immunodeficiency virus (HIV) in vitro, significantly suppressed both the infectivity and the cytopathic effect of the virus, with 2',3'-dideoxycytidine (ddCyd) being the most potent of the series (total antiviral protection at 0.5-1.0 microM). We have compared three factors likely to be of significance in determining the pharmacological activity of these compounds, i.e., (i) their abilities to influence pool sizes of physiological deoxynucleoside-5'-triphosphates, (ii) their capacity to generate the corresponding 2',3'-dideoxynucleoside-5'-triphosphates, and (iii) the effectiveness of these nucleoside-5'-triphosphates as inhibitors of HIV reverse transcriptase. In MOLT-4 cells (a human T cell line), ddCyd was the compound most efficiently converted to its 5'-triphosphate, whereas 2',3'-dideoxyguanosine and 2',3'-dideoxythymidine were the compounds least efficiently converted, generating levels of their corresponding 5'-triphosphates less than 0.1% of that seen with ddCyd when these nucleosides were compared on an equimolar basis (5 microM). The 3'-azido analogue of 2',3'-dideoxythymidine fell intermediate between these two extremes. As inhibitors of HIV reverse transcriptase, however, all the 5'-triphosphates, with the exception of 2',3'-dideoxyinosine-5'-triphosphate, fell within a narrow range of activity (Ki, 0.10-0.26 microM), affinities some 40-60 fold greater than those of the corresponding physiological 2'-deoxynucleoside-5'-triphosphates. Significant alterations in pool sizes of physiological 2'-deoxynucleoside-5'-triphosphates were not observed at pharmacologically effective drug levels. The relative ability of 2',3'-dideoxynucleosides to generate 5'-triphosphates intracellularly thus correlates much more closely than do the other two factors examined, in capacity to block HIV replication. These studies support the conclusion that, for purposes of design of new compounds of this general class, factors influencing efficiency of nucleotide formation and degradation (e.g., membrane transport mechanisms, affinities for nucleoside kinases and for nucleotide kinases and phosphatases) may be of equal or even greater importance than differences in the relative abilities of the resultant 2',3'-dideoxynucleoside-5'-triphosphates to inhibit the viral reverse transcriptase.

DNA replication fidelity: kinetics and thermodynamics.

Mechanisms that control the fidelity of DNA replication are discussed. Data are reviewed for 3 steps in a fidelity pathway: nucleotide insertion, exonucleolytic proofreading, and extension from matched and mismatched 3'-primer termini. Fidelity mechanisms that involve predominantly Km discrimination, Vmax discrimination, or a combination of the two are analyzed in the context of a simple model for fidelity. Each fidelity step is divided into 2 components, thermodynamic and kinetic. The thermodynamic component, which relates to free-energy differences between right and wrong base pairs, is associated with a Km discrimination mechanism for polymerase. The kinetic component, which represents the enzyme's ability to select bases for insertion and excision to achieve fidelity greater than that available from base pairing free-energy differences, is associated with a Vmax discrimination mechanism for polymerase. Currently available fidelity data for nucleotide insertion and primer extension in the absence of proofreading appears to have relatively large Km and small Vmax components. An important complication can arise when analyzing data from polymerases containing an associated 3'-exonuclease activity. In the presence of proofreading, a Vmax discrimination mechanism is likely to occur, but this may be the result of two Km discrimination mechanisms acting serially, one for nucleotide insertion and the other for excision. Possible relationships between base pairing free energy differences measured in aqueous solution and those defined within the polymerase active cleft are considered in the context of the enzyme's ability to exclude water, at least partially, from the vicinity of its active site.

[Control mechanisms of the ribonucleotide reduction in mammalian tissue (author's transl)]. / Steuermechanismen der Ribonucleotid-Reduktion in Säugetiergewebe.

The ribonucleoside diphosphate reductase (E.C.1.17.4.1) was partially purified from Ehrlich ascites carcinoma and regenerating rat liver. The specific activity of the two enzyme preparations did not vary with regard to the reduction of UDP, ADP, CDP and GDP. Similarly the regulation of the enzyme system by deoxyribonucleoside triphosphate (dNTP) is almost identical. Through the application of deoxyribonucleosides (10(-3) or 2 x 10(-3)M) and measurement of the dNTP content it was found in Ehrlich and Yoshida ascites tumours that these control mechanisms are transmissible to whole cells. dATP inhibits the reduction of all four nucleoside diphosphates. dTTP stimulates the reduction of GDP, dCTP that of UDP and dGTP that of ADP.