Thioredoxin and glutaredoxin-mediated redox regulation of ribonucleotide reductase Sengupta R,Holmgren A简

Ribonucleotide reductase (RNR), the rate-limiting enzyme in DNA synthesis, catalyzes reduction of the different ribonucleotides to their corresponding deoxyribonucleotides. The crucial role of RNR in DNA synthesis has made it an important target for the development of antiviral and anticancer drugs. Taking account of the recent developments in this field of research, this review focuses on the role of thioredoxin and glutaredoxin systems in the redox reactions of the RNR catalysis.     

Enzymatic removal of ribonucleotides from DNA is essential for mammalian genome integrity and development

The presence of ribonucleotides in genomic DNA is undesirable given their increased susceptibility to hydrolysis. Ribonuclease (RNase) H enzymes that recognize and process such embedded ribonucleotides are present in all domains of life. However, in unicellular organisms such as budding yeast, they are not required for viability or even efficient cellular proliferation, while in humans, RNase H2 hypomorphic mutations cause the neuroinflammatory disorder Aicardi-Goutières syndrome. Here, we report that RNase H2 is an essential enzyme in mice, required for embryonic growth from gastrulation onward. RNase H2 null embryos accumulate large numbers of single (or di-) ribonucleotides embedded in their genomic DNA (>1,000,000 per cell), resulting in genome instability and a p53-dependent DNA-damage response. Our findings establish RNase H2 as a key mammalian genome surveillance enzyme required for ribonucleotide removal and demonstrate that ribonucleotides are the most commonly occurring endogenous nucleotide base lesion in replicating cells.     

Ribonucleotides in DNA: Origins,repair and consequences

While primordial life is thought to have been RNA-based (Cech, Cold Spring Harbor Perspect. Biol. 4 (2012) a006742), all living organisms store genetic information in DNA, which is chemically more stable. Distinctions between the RNA and DNA worlds and our views of “DNA” synthesis continue to evolve as new details emerge on the incorporation, repair and biological effects of ribonucleotides in DNA genomes of organisms from bacteria through humans.     

Allosteric regulation of the class III anaerobic ribonucleotide reductase from bacteriophage T4

Ribonucleotide reductase (RNR) is an essential enzyme in all organisms. It provides precursors for DNA synthesis by reducing all four ribonucleotides to deoxyribonucleotides. The overall activity and the substrate specificity of RNR are allosterically regulated by deoxyribonucleoside triphosphates and ATP, thereby providing balanced dNTP pools. We have characterized the allosteric regulation of the class III RNR from bacteriophage T4. Our results show that the T4 enzyme has a single type of allosteric site to which dGTP, dTTP, dATP, and ATP bind competitively. The dissociation constants are in the micromolar range, except for ATP, which has a dissociation constant in the millimolar range. ATP and dATP are positive effectors for CTP reduction, dGTP is a positive effector for ATP reduction, and dTTP is a positive effector for GTP reduction. dATP is not a general negative allosteric effector. These effects are similar to the allosteric regulation of class Ib and class II RNRs, and to the class Ia RNR of bacteriophage T4, but differ from that of the class III RNRs from the host bacterium Escherichia coli and from Lactococcus lactis. The relative rate of reduction of the four substrates was measured simultaneously in a mixed-substrate assay, which mimics the physiological situation and illustrates the interplay between the different effectors in vivo. Surprisingly, we did not observe any significant UTP reduction under the conditions used. Balancing of the pyrimidine deoxyribonucleotide pools may be achieved via the dCMP deaminase and dCMP hydroxymethylase pathways.     

Ribonucleotide reductases: Substrate specificity by allostery

Ribonucleotide reductases catalyze in all living organisms the production of deoxynucleotides from ribonucleotides. A single enzyme provides a balanced supply of the four dNTPs required for DNA replication. Three different but related classes of enzymes are known. Each class catalyzes the same chemistry using a common radical mechanism involving a thiyl radical of the enzyme but the three classes employ different mechanisms for the generation of the radical. For each class a common allosteric mechanism with ATP and dNTPs as effectors directs the substrate specificity of the enzymes ensuring the appropriate balance of the four dNTPs for DNA replication. Recent crystallographic studies of the catalytic subunits from each class in combination with allosteric effectors, with and without cognate substrates, delineated the structural changes caused by effector binding that direct the specificity of the enzymes towards reduction of the appropriate substrate.     

Differential Activities of DNA Polymerases in Processing Ribonucleotides during DNA Synthesis in Archaea

Consistent with the fact that ribonucleotides (rNTPs) are in excess over deoxyribonucleotides (dNTPs) in vivo, recent findings indicate that replicative DNA polymerases (DNA Pols) are able to insert ribonucleotides (rNMPs) during DNA synthesis, raising crucial questions about the fidelity of DNA replication in both Bacteria and Eukarya. Here, we report that the level of rNTPs is 20-fold higher than that of dNTPs in Pyrococcus abyssi cells. Using dNTP and rNTP concentrations present in vivo, we recorded rNMP incorporation in a template-specific manner during in vitro synthesis, with the family-D DNA Pol (PolD) having the highest propensity compared with the family-B DNA Pol and the p41/p46 complex. We also showed that ribonucleotides accumulate at a relatively high frequency in the genome of wild-type Thermococcales cells, and this frequency significantly increases upon deletion of RNase HII, the major enzyme responsible for the removal of RNA from DNA. Because ribonucleotides remain in genomic DNA, we then analyzed the effects on polymerization activities by the three DNA Pols. Depending on the identity of the base and the sequence context, all three DNA Pols bypass rNMP-containing DNA templates with variable efficiency and nucleotide (mis)incorporation ability. Unexpectedly, we found that PolD correctly base-paired a single ribonucleotide opposite rNMP-containing DNA templates. An evolutionary scenario is discussed concerning rNMP incorporation into DNA and genome stability.     

How the misincorporation of ribonucleotides into genomic DNA can be both harmful and helpful to cells

Ribonucleotides are misincorporated into replicating DNA due to the similarity of deoxyribonucleotides and ribonucleotides, the high concentration of ribonucleotides in the nucleus and the imperfect accuracy of replicative DNA polymerases in choosing the base with the correct sugar. Embedded ribonucleotides change certain properties of the DNA and can interfere with normal DNA transactions. Therefore, misincorporated ribonucleotides are targeted by the cell for removal. Failure to remove ribonucleotides from DNA results in an increase in genome instability, a phenomenon that has been characterized in various systems using multiple assays. Recently, however, another side to ribonucleotide misincorporation has emerged, where there is evidence for a functional role of misinserted ribonucleotides in DNA, leading to beneficial consequences for the cell. This review examines examples of both positive and negative effects of genomic ribonucleotide misincorporation in various organisms, aiming to highlight the diversity and the utility of this common replication variation.     

The role of RNA primer in discontinuous DNA replication in isolated nuclei from HeLa cells

RNA-primed discontinuous DNA synthesis was studied in an in vitro system consisting of washed nuclei from synchronized S-phase HeLa cells. A new technique proved useful for the purification of short nascent fragments of DNA (Okazaki fragments). Mercurated dCTP was substituted for dCTP in the DNA synthesis reaction. Short nascent pieces (4–6 S) of mercurated DNA were found to bind preferentially to sulfhydryl-agarose, and could be eluted with mercaptoethanol. The isolated fragments were assayed for the presence of covalently linked RNA by the spleen exonuclease method described by Kurosawa et al. (Kurosawa, Y., Ogawa, T., Hirose, S., Okazaki, T. and Okazaki, R. (1975) J. Mol. Biol. 96, 653–664). Following a 30 s incubation with [³H]TTP in the absence of added ribonucleotides, approximately 20% of the nascent strands synthesized in washed nuclear preparations had RNA attached. These RNA primers either preexisted in the nuclei or were formed from endogenous ribonucleotides. The 5′ ends of the primers appeared to be largely in a phosphorylated state. In the absence of added ribonucleotides, these RNA-DNA linkages disappeared within 2 min, whereas if ribonucleotides were added, the number of RNA primers increased to 40% and remained at this level for greater than 2 min. To obtain maximal levels of RNA primer, the addition of all three of the ribonucleotides, rCTP, rGTP and rUTP (0.1 mM), as well as high levels of rATP (5 mM) was required. Addition of ribonucleotides also markedly enhanced the amount of nascent DNA fragments synthesized. However, in the absence of added ribonucleotides, after RNA primers had disappeared, nascent DNA fragments were still initiated at a significant rate. These results suggest that RNA primers play an important role in the initiation of Okazaki fragments but that synthesis can also be initiated by alternative mechanisms. An important role for ATP in RNA primer synthesis is suggested.     

Cloning and characterization of ribonucleotide reductase from Chlamydia trachomatis

In all organisms the deoxyribonucleotide precursors required for DNA synthesis are synthesized from ribonucleotides, a reaction catalyzed by ribonucleotide reductase. In a previous study we showed that Chlamydia trachomatis growth was inhibited by hydroxyurea, an inhibitor of ribonucleotide reductase, and a mutant resistant to the cytotoxic effects of the drug was isolated. Here we report the cloning, expression, and purification of the R1 and R2 subunits of the C. trachomatis ribonucleotide reductase. In comparison with other ribonucleotide reductases, the primary sequence of protein R1 has an extended amino terminus, and the R2 protein has a phenylalanine where the essential tyrosine is normally located. Despite its unusual primary structure, the recombinant enzyme catalyzes the reduction of CDP to dCDP. Results from deletion mutagenesis experiments indicate that while the extended amino terminus of the R1 protein is not required for enzyme activity, it is needed for allosteric inhibition mediated by dATP. Results with site-directed mutants of protein R2 suggest that the essential tyrosine is situated two amino acids downstream of its normal location. Finally, Western blot analysis show that the hydroxyurea-resistant mutant C. trachomatis isolate overexpresses both subunits of ribonucleotide reductase. At the genetic level, compared with wild type C. trachomatis, the resistant isolate has a single base mutation just upstream of the ATG start codon of the R2 protein. The possibility that this mutation affects translational efficiency is discussed.     

Purine metabolism in Trypanosoma brucei gambiense

The procyclic forms of Trypanosoma brucei gambiense do not incorporate glycine or serine into ribonucleotides. Although de novo purine synthesis does not occur, all purine bases and ribonucleotides are interconverted, indicating the presence of active salvage pathways. Guanine is actively deaminated to xanthine by guanase activity. Purine ribonucleosides are cleaved to their respective free bases. The order of salvage efficiency for purine bases and their respective ribonucleotides is: adenine > hypoxanthine > guanine > xanthine.     

Thioredoxin and glutaredoxin-mediated redox regulation of ribonucleotide reductase

Ribonucleotide reductase(RNR), the rate-limitingenzyme in DNA synthesis, catalyzes reduction of thedifferent ribonucleotides to their corresponding deoxyri-bonucleotides. The crucial role of RNR in DNA synthesishas made it an important target for the development ofantiviral and anticancer drugs. Taking account of the re-cent developments in this field of research, this reviewfocuses on the role of thioredoxin and glutaredoxin sys-tems in the redox reactions of the RNR catalysis.     

Structure and function of the radical enzyme ribonucleotide reductase

Ribonucleotide reductases (RNRs) catalyze all new production in nature of deoxyribonucleotides for DNA synthesis by reducing the corresponding ribonucleotides. The reaction involves the action of a radical that is produced differently for different classes of the enzyme. Class I enzymes, which are present in eukaryotes and microorganisms, use an iron center to produce a stable tyrosyl radical that is stored in one of the subunits of the enzyme. The other classes are only present in microorganisms. Class II enzymes use cobalamin for radical generation and class III enzymes, which are found only in anaerobic organisms, use a glycyl radical. The reductase activity is in all three classes contained in enzyme subunits that have similar structures containing active site cysteines. The initiation of the reaction by removal of the 3′-hydrogen of the ribose by a transient cysteinyl radical is a common feature of the different classes of RNR. This cysteine is in all RNRs located on the tip of a finger loop inserted into the center of a special barrel structure. A wealth of structural and functional information on the class I and class III enzymes can now give detailed views on how these enzymes perform their task. The class I enzymes demonstrate a sophisticated pattern as to how the free radical is used in the reaction, in that it is only delivered to the active site at exactly the right moment. RNRs are also allosterically regulated, for which the structural molecular background is now starting to be revealed.     

Purine metabolism in the bloodstream forms of trypanosoma gambiense and Trypanosoma rhodesiense

Bloodstream forms of Trypanosoma brucie gambiense and Trypanosoma brucei rhodesiense are incapable of de novo purine synthesis. Purine bases are converted directly to ribonucleotides and with the exception of guanine, are stable. Guanine is incorporated directly into ribonucleotides and also deaminated to xanthine. Purine ribonucleosides are hydrolyzed rapidly; these reactions may limit their incorporation since purine bases label the nucleotide pools more efficiently than do ribonucleosides. The apparent order of salvage efficiency for ribonucleosides is adenosine>inosine>guanosine>xanthosine for both organisms. T. b. gambiense salvages purine bases in the same order, while T. b. rhodesiense salvages purine bases in the order hypoxanthine>adenine>guanine>xanthine.     

Replication of ribonucleotide-containing DNA templates by yeast replicative polymerases

The major replicative DNA polymerases of S. cerevisiae (Pols α, δ, and ?) incorporate substantial numbers of ribonucleotides into DNA during DNA synthesis. When these ribonucleotides are not removed in vivo, they reside in the template strand used for the next round of replication and could potentially reduce replication efficiency and fidelity. To examine if the presence of ribonucleotides in a DNA template impede DNA synthesis, we determined the efficiency with which Pols α, δ, and ? copy DNA templates containing a single ribonucleotide. All three polymerases can replicate past ribonucleotides. Relative to all-DNA templates, bypass of ribo-containing templates is slightly reduced, to extents that depend on the identity of the ribo and the sequence context in which it resides. Bypass efficiencies for Pols δ and ? were increased by increasing the dNTP concentrations to those induced by cellular stress, and in the case of Pol ?, by inactivating the 3'-exonuclease activity. Overall, ribonucleotide bypass efficiencies are comparable to, and usually exceed, those for the common oxidative stress-induced lesion 8-oxo-guanine.     

DNA replication by bacteriophage T4 proteins. The T4 43, 32, 44--62, And 45 proteins are required for strand displacement synthesis at nicks in duplex DNA.

The T4 bacteriophage gene 43 (T4 DNA polymerase), 32 (DNA helix-destabilizing protein), and 45 proteins and the complex of the gene 44 and 62 proteins are all required for DNA synthesis beginning at single-stranded breaks in duplex DNA. This synthesis occurs by strand displacement and is not dependent on ribonucleotides, the T4 gene 41 protein, or the T4 initiating protein, each of which is required to begin new chains on single-stranded templates. Electron microscopic analysis shows that duplex molecules with long single-stranded branches are the predominant products of this strand displacement synthesis.     

A docking model of human ribonucleotide reductase with flavin and phenosafranine

Ribonucleotide Reductase (RNR) is an enzyme responsible for the reduction of ribonucleotides to their corresponding Deoxyribonucleotides (DNA), which is a building block for DNA replication and repair mechanisms. The key role of RNR in DNA synthesis and control in cell growth has made this an important target for anticancer therapy. Increased RNR activity has been associated with malignant transformation and tumor cell growth. In recent years, several RNR inhibitors, including Triapine, Gemcitabine and GTI-2040, have entered the clinical trials. Our current work focuses on an attempted to dock this inhibitors Flavin and Phenosafranine to curtail the action of human RNR2. The docked inhibitor Flavin and Phenosafranine binds at the active site with THR176, which are essential for free radical formation. The inhibitor must be a radical scavenger to destroy the tyrosyl radical or iron metal scavenger. The iron or radical site of R2 protein can react with one-electron reductants, whereby the tyrosyl radical is converted to a normal tyrosine residue. However, compounds such as Flavin and Phenosafranine were used in most of the cases to reduce the radical activity. The docking study was performed for the crystal structure of human RNR with the radical scavengers Flavin and Phenosafranine to inhibit the human RNR2. This helps to understand the functional aspects and also aids in the development of novel inhibitors for the human RNR2.     

Multiple amino acid substitutions allow DNA polymerases to synthesize RNA

DNA and RNA polymerase exhibit similarities in structures and catalytic mechanisms, suggesting that both classes of enzymes are evolutionarily related. To probe the biochemical and structure-function relationship between the two classes of polymerases, a large library (200,000 members) of mutant Thermus aquaticus DNA polymerase I (Taq pol I) was created containing random substitutions within a portion of the dNTP binding site (motif A; amino acids 605-617), and a fraction of all selected active Taq pol I (291 of 8000) was tested for the ability to incorporate successive ribonucleotides; 23 unique mutants that added rNTPs into a growing polynucleotide chain were identified and sequenced. These mutants, each containing one to four substitutions, incorporate ribonucleotides at a efficiency approaching 10(3)-fold greater than that of wild type Taq pol I. Several mutants added successive ribonucleotides and thus can catalyze the synthesis of RNA. Sequence analysis of these mutants demonstrates that at least two amino acid residues are involved in excluding ribonucleotides from the active site. Interestingly, wild type DNA polymerases from several distinct families selectively discriminate against rUTP. This study suggests that current DNA and RNA polymerases could have evolved by divergent evolution from an ancestor that shared a common mechanism for polynucleotide synthesis.