1). The effect of temperature, P450 inhibitors (pyrazole and imidazole), sulphydryl reagents (iodoacetamide and N-ethyl maleimide) and glutathione on the activation of CCl4 in rat liver microsomes was studied. Spin trapping of CCI3', covalent binding of CCl4 to protein and CCl4-dependent MDA formation were used as indices of CCl4 metabolism. Formation of PBN-CCI3' adduct, 14CCl4 covalent binding to protein and CCl4-dependent :MDA production were dependent on temperature range from 15-40°C. The transition temperature was at 26.7 -27 .5°C when the activation was measured by formation of PBNCCl3' adduct and specific 14CCl4 covalent binding. The transition temperature was found to be 34.3°C when CCl4 -dependent MDA production was taken as the index of the activation of CCI4. Pyrazole, imidazole and iodoacetamide inhibited CC14 -dependent MDA formation only at high concentrations (10-20 mM), whereas glutathione showed a strong inhibitory effect on CCl4-stimulated lipid peroxidation. MDA formation was nearly 100°;6 inhibited by 1 roM GSH. GSH also delayed the onset of lipid peroxidation. N-ethyl maleimide (NEM) exerted biphasic effects on CCl4 -dependent MDA formation. The lower concentration of NEM (0.5 mM-l mM) reduced the :MDA prodUction, while the higher concentration of NEM (5-10 mM) enhanced the MDA formation. 2). Ribonucleotide reductase was partially purified from juvenile normal rat liver. The enzyme was purified 30 fold after DEAE-cellulose chromatography. The CDP reductase activity in tissues with different growth states or rates was compared. The enzyme activity was developed well in juvenile rat liver, regenerating liver and hepatoma (cells), while the enzyme activity was undetectable in adult rat liver and sham-operated rat liver. The enzyme activity in Yoshida cells was 3-fold of the activity in Morris 5123tc tumours. Dithiothreitol (DIT) activated the activity of CDP reductase from 48h and 60h regenerating liver, but DIT did not activate the enzyme activity of juvenile 'normal rat liver. The possible mechanism of the activation of enzyme activity by DIT was discussed and a mechanism of regulation of the ribonucleotide reductase activity in regenerating liver was suggested. 3). The effect of the lipid peroxidation product 4-hydroxynonenal (HNE) on CDP reductase from juvenile normal rat liver was investigated. HNE inhibited the CDP reductase activity. The inhibition was dependent on the concentration of HNE and the incubation time. The enzyme activity was reduced 500/0 by 0.1 roM HNE. The inhibitory effect of HNE was irreversible. DIT protected the enzyme against HNE suggesting that HNE inhibited the activity of ribonucleotide reductase from rat liver through the mechanism of blockage of functional SH groups in the enzyme protein.

Trypanosoma brucei causes African sleeping sickness in humans and Nagana in cattle. There are no vaccines available against the disease and the current treatment is also not satisfactory because of inefficacy and numerous side effects of the used drugs. T. brucei lacks de novo synthesis of purine nucleosides; hence it depends on the host to make its purine nucleotides. T. brucei has a high affinity adenosine kinase (TbAK), which phosphorylates adenosine, deoxyadenosine (dAdo), inosine and their analogs. RNAi experiments confirmed that TbAK is responsible for the salvage of dAdo and the toxicity of its substrate analogs. Cell growth assays with the dAdo analogs, Ara-A and F-Ara-A, suggested that TbAK could be exploited for drug development against the disease. It has previously been shown that when T. brucei cells were cultivated in the presence of 1 mM deoxyadenosine (dAdo), they showed accumulation of dATP and depletion of ATP nucleotides. The altered nucleotide levels were toxic to the trypanosomes. However the salvage of dAdo in trypanosomes was dramatically reduced below 0.5 mM dAdo. Radiolabeled dAdo experiments showed that it (especially at low concentrations) is cleaved to adenine and converted to ATP. The recombinant methylthioadenosine phosphorylase (TbMTAP) cleaved methylthioadenosine, dAdo and adenosine into adenine and sugar-1-P in a phosphate-dependent manner. The trypanosomes became more sensitive to dAdo when TbMTAP was down-regulated in RNAi experiments. The RNAi experiments confirmed that trypanosomes avoid dATP accumulation by cleaving dAdo. The TbMTAP cleavage-resistant nucleoside analogs, FANA-A and Ara-A, successfully cured T. brucei-infected mice. The DNA building block dTTP can be synthesized either via thymidylate synthase in the de novo pathway or via thymidine kinase (TK) by salvage synthesis. We found that T. brucei and three other parasites contain a tandem TK where the gene sequence was repeated twice or four times in a single open reading frame. The recombinant T. brucei TK, which belongs to the TK1 family, showed broad substrate specificity. The enzyme phosphorylated the pyrimidine nucleosides thymidine and deoxyuridine, as well as the purine nucleosides deoxyinosine and deoxyguanosine. When the repeated sequences of the tandem TbTK were expressed individually as domains, only domain 2 was active. However, the protein could not dimerize and had a 5-fold reduced affinity to its pyrimidine substrates but a similar turnover number as the full-length enzyme. The expressed domain 1 was inactive and sequence analysis revealed that some active residues, which are needed for substrate binding and catalysis, are absent. Generally, the TK1 family enzymes form dimers or tetramers and the quaternary structure is linked to the affinity for the substrates. The covalently linked inactive domain-1 helps domain-2 to form a pseudodimer for the efficient binding of substrates. In addition, we discovered a repetition of an 89-bp sequence in both domain 1 and domain 2, which suggests a genetic exchange between the two domains. T. brucei is very dependent on de novo synthesis via ribonucleotide reductase (RNR) for the production of dNTPs. Even though T. brucei RNR belongs to the class Ia RNR family and contains an ATP-binding cone, it lacks inhibition by dATP. The mechanism behind the RNR activation by ATP and inactivation by dATP was a puzzle for a long time in the ~50 years of RNR research. We carried out oligomerization studies on mouse and E. coli RNRs, which belongs to the same family as T. brucei, to get an understanding of the molecular mechanism behind overall activity regulation. We found that the oligomerization status of RNRs and overall activity mechanism are interlinked with each other. / Targeting the nucleotide metabolism of the mammalian pathogen Trypanosoma brucei.

Streptococcus sanguinis is the second most common causative agent of bacterial infective endocarditis (IE). Risk of S. sanguinis IE is dependent on pre-disposing damage to the heart valve endothelium, which results in deposition of clotting factors for formation of a sterile thrombus (referred to as vegetation). Despite medical advances, high mortality and morbidity rates persist. Molecular characterization of S. sanguinis virulence determinants may enable development of prevention methods. In a previous screen for S. sanguinis virulence determinants by signature-tagged mutagenesis (STM) an attenuated mutant was identified with a transposon insertion in the nrdD gene, encoding an anaerobic ribonucleotide reductase. Evaluation of this mutant, as well as an nrdD in-frame deletion mutant, JFP27, by a soft-agar growth assay confirmed the anaerobic growth sensitivity of these strains. These studies suggest that an oxygen gradient occurs at the site of infection which selects for expression of anaerobic-specific genes at the nexus of the vegetation. The random STM screen failed to identify any favorable streptococcal surface-exposed prophylactic candidates. It was also apparent that additional genetic tools were required to facilitate the in vivo analyses of mutant strains. As it was desirable to insert antibiotic resistance markers into the chromosome, we identified a chromosomal site for ectopic expression of foreign genes. In vitro and in vivo analyses verified that insertion into this site did not affect important cellular phenotypes. The genetic tools developed facilitated further in vivo screening of S. sanguinis cell wall-associated (Cwa) protein mutants. A directed application of STM was employed for a comprehensive analysis of this surface protein class in the rabbit model of IE. Putative sortases, upon which Cwa proteins are dependent for cell surface localization, were also evaluated. No single S. sanguinis Cwa protein was determined essential for IE by STM screening; however competitiveness for colonization of the infection site was reduced for the mutant lacking expression of sortase A. The studies described here present a progressive picture of S. sanguinis IE, beginning with surface protein-dependent colonization of the vegetation in early IE, that later shifts to a bacterial persistence in situ dependent on condition-specific housekeeping genes, including nrdD.

<p>Diiron carboxylate proteins constitute an important class of metall-containing enzymes. These proteins perform a multitude of reactions in biological systems that normally involve activation of molecular oxygen at the diiron site.</p><p>During activation and functioning of these proteins their diiron sites undergo redox changes in a rather wide range: from diferrous (FeII-FeII) to high potential intermediate Q(FeIV-FeIV). Two of these redox states are paramagnetic: (FeIV-FeIII), called high potential intermediate X, and (FeII-FeIII), called mixed-valent state of the diiron carboxylate proteins. In the present work it has been shown that these redox states are of functional relevance in two proteins with different functions.</p><p>Ribonucleotide reductase (RNR) from the human parasite<i> Chlamydia trachomatis</i> is a class I RNR. It is typical for class I RNR to initiate the enzymatic reaction on its large subunit, protein R1, by activation from a stable tyrosyl free radical in its small subunit, protein R2. This radical, in its turn, is formed through oxygen activation by the diiron center. In C. trachomatis the tyrosine residue is replaced by phenylalanine, which cannot form a radical. We have shown in the present work, that active <i>C. trachomatis</i> RNR uses the FeIII-FeIV state of the diiron carboxylate cluster in R2 instead of a tyrosyl radical to initiate the catalytic reaction.</p><p>The alternative oxidase (AOX) is a ubiquinol oxidase found in the mitochondrial respiratory chain of plants. The existence of the diiron carboxylate center in this protein was predicted on the basis of a conserved sequence motif consisting of the proposed iron ligands, four glutamate and two histidine residues. In experiments modeling the conditions of the enzyme catalytic cycle, i.e. reduction and reoxygenation of the overexpressed AOX in <i>Escherichia coli</i> membranes we were able to generate an EPR signal characteristic of a mixed-valent Fe(II)/Fe(III) binuclear iron center. The alternative oxidase is the first membrane protein where the existence of the diiron carboxylate center has been shown experimentally.</p>

A prerequisite for a multicellular organism to survive is the ability to correctly replicate and repair DNA while minimizing the number of heritable mutations. To achieve this, cells need a balanced supply of deoxyribonucleoside triphosphates (dNTPs), the precursors for DNA synthesis. The rate-limiting step in de novo biosynthesis of dNTPs is catalyzed by the enzyme ribonucleotide reductase (RNR). The classic eukaryotic RNR enzyme consists of a large and a small subunit. Together, these subunits form a heterotetrameric RNR complex. The larger subunit harbours active sites whereas the smaller subunit contains a stable tyrosyl free radical. Both subunits are required for RNR activity. Since failure to correctly regulate de novo dNTP biosynthesis can lead to misincorporation of nucleotides into DNA, genetic abnormalities and cell death, RNR activity is tightly regulated. The regulation of RNR activity involves cell cycle-specific expression and degradation of the RNR proteins, as well as binding of allosteric effectors to the large RNR subunit. In this thesis, in vitro assays based on purified recombinant RNR proteins, in combination with in vivo assays, have been used successfully to study the regulation of RNR activity in response to DNA damage. I present new findings regarding the function of an alternative mammalian RNR small subunit, and on the role of a small RNR inhibitor protein of fission yeast, during normal growth and after DNA damage. I also show conclusively that there are fundamental differences in the regulation of dNTP biosynthesis between the cells of higher and lower eukaryotes after DNA damage.

Diiron carboxylate proteins constitute an important class of metall-containing enzymes. These proteins perform a multitude of reactions in biological systems that normally involve activation of molecular oxygen at the diiron site. During activation and functioning of these proteins their diiron sites undergo redox changes in a rather wide range: from diferrous (FeII-FeII) to high potential intermediate Q(FeIV-FeIV). Two of these redox states are paramagnetic: (FeIV-FeIII), called high potential intermediate X, and (FeII-FeIII), called mixed-valent state of the diiron carboxylate proteins. In the present work it has been shown that these redox states are of functional relevance in two proteins with different functions. Ribonucleotide reductase (RNR) from the human parasite Chlamydia trachomatis is a class I RNR. It is typical for class I RNR to initiate the enzymatic reaction on its large subunit, protein R1, by activation from a stable tyrosyl free radical in its small subunit, protein R2. This radical, in its turn, is formed through oxygen activation by the diiron center. In C. trachomatis the tyrosine residue is replaced by phenylalanine, which cannot form a radical. We have shown in the present work, that active C. trachomatis RNR uses the FeIII-FeIV state of the diiron carboxylate cluster in R2 instead of a tyrosyl radical to initiate the catalytic reaction. The alternative oxidase (AOX) is a ubiquinol oxidase found in the mitochondrial respiratory chain of plants. The existence of the diiron carboxylate center in this protein was predicted on the basis of a conserved sequence motif consisting of the proposed iron ligands, four glutamate and two histidine residues. In experiments modeling the conditions of the enzyme catalytic cycle, i.e. reduction and reoxygenation of the overexpressed AOX in Escherichia coli membranes we were able to generate an EPR signal characteristic of a mixed-valent Fe(II)/Fe(III) binuclear iron center. The alternative oxidase is the first membrane protein where the existence of the diiron carboxylate center has been shown experimentally.

Ribonucleotide reductase is a remarkable enzyme that catalyzes the rate-limiting step in the synthesis of the 2'-deoxynucleoside triphosphates. The intent of this project was to characterize the ribonucleotide reductase encoded by the orthopoxvirus, vaccinia. The first objective was to study the structural and functional features of the viral small subunit protein of ribonucleotide reductase. The viral reductase gene was engineered into an expression vector and expressed in Escherichia coli. The purified recombinant protein was then characterized and compared with other ribonucleotide reductase small subunits from different organisms. The physical characteristics of the vaccinia virus enzyme showed a strong similarity to the features of the mammalian counterpart. A second aim of this project was to establish the transcriptional and translational kinetics of ribonucleotide reductase gene expression during the time course of viral infection in cultured mammalian cells. In addition, the activity and stability of the enzyme in the viral system was measured and the accumulation of ribonucleotide reductase protein was quantitated. By also quantitating the accumulation of viral DNA synthesis, a direct comparison can be made between the the synthesis and utilization of deoxynucleotide precursors. A third objective of this work was to detail the mechanism by which hydroxyurea inactivates the vaccinia virus ribonucleotide reductase. Visible spectroscopy and electron paramagnetic resonance spectroscopy clearly demonstrated that the inhibitor destroys the free radical moiety in the viral small subunit protein. In addition, in vivo studies revealed that inhibition by hydroxyurea can be circumvented during viral infection. The exogenous addition of deoxyadenosine reversed the block to viral growth that was imposed by hydroxyurea, and stabilized hydroxyurea induced deoxynucleotide pool imbalances. These inhibition studies suggest that there may be a differential sensitivity of the enzyme towards hydroxyurea in the presence of various substrates. / Graduation date: 1993

In order to build any structure, a good supply of materials, accurate workers and quality control are needed. This is even the case when constructing DNA, the so-called “Code of Life.” For a species to continue to exist, this DNA code must be copied with incredibly high accuracy when each and every cell replicates. In fact, just one mistake in the 12 million bases that comprise the genome of budding yeast, Saccharomyces cerevisiae, can be fatal. DNA is composed of a double strand helix made up of just four different bases repeated millions of times. The building blocks of DNA are the deoxyribonucleotides (dNTPs); dCTP, dTTP, dATP and dGTP. Their production and balance are carefully controlled within each cell, largely by the key enzyme Ribonucleotide Reductase (RNR). Here, we studied how the enzymes that copy DNA, the replicative polymerases α, δ and ε, cope with the effects of an altered dNTP pool balance. An introduced mutation in the allosteric specificity site of RNR in a strain of S. cerevisiae, rnr1-Y285A, leads to elevated dCTP and dTTP levels and has been shown to have a 14-fold increase in mutation rate compared to wild type. To ascertain the full effects of the dNTP pool imbalance upon the replicative polymerases, we disabled one of the major quality control systems in a cell that corrects replication errors, the post-replicative Mismatch Repair system. Using both the CAN1 reporter assay and whole genome sequencing, we found that, despite inherent differences between the polymerases, their replication fidelity was affected very similarly by this dNTP pool imbalance. Hence, the high dCTP and dTTP forced Pol ε and Pol α/δ to make the same mistakes. In addition, the mismatch repair machinery was found to correct replication errors driven by this dNTP pool imbalance with highly variable efficiencies. Another mechanism to protect cells from DNA damage during replication is a checkpoint that can be activated to delay the cell cycle and activate repair mechanisms. In yeast, Mec1 and Rad53 (human ATR and Chk1/Chk2) are two key S-phase checkpoint proteins. They are essential as they are also required for normal DNA replication and dNTP pool regulation. However the reason why they are essential is not well understood. We investigated this by mutating RAD53 and analyzing dNTP pools and gene interactions. We show that Rad53 is essential in S-phase due to its role in regulating basal dNTP levels by action in the Dun1 pathway that regulates RNR and Rad53’s compensatory kinase function if dNTP levels are perturbed. In conclusion we present further evidence of the importance of dNTP pools in the maintenance of genome integrity and shed more light on the complex regulation of dNTP levels.

Deoxyribonucleic acid (DNA) carries all the genetic information of a cell. Ribonucleotide reductase (RNR) provides balanced pools of all four dNTPs, the building blocks of DNA. These building blocks are needed during DNA synthesis and repair. A failure in the control of the dNTP levels and/or their relative amounts leads to cell death or genetic abnormalities. Because of its central role in dNTP metabolism, RNR is highly regulated on multiple levels. The active RNR enzyme consists of two non-identical subunits called proteins R1 and R2. In mammalian cells, during an unperturbed cell cycle, the activity of RNR is highest during S and G2 phases. This is achieved by de novo synthesis of the limiting R2 protein at the onset of S phase, and by controlled degradation of the R2 protein during mitosis. This thesis deals with both the S phase-specific transcription of the mouse R2 gene, and the M phase-specific degradation of the mouse R2 protein. Sequence comparison of the mouse R2 promoter to human and guinea pig R2 promoters revealed some conserved elements. These putative regulatory elements were tested in both in vitro and in vivo transcription assays. We demonstrated that the previously identified, NF-Y binding CCAAT box is essential for high-level expression from the R2 promoter, but not for its S phase specificity. In addition, the conserved TATA box is dispensable both for basal and S phase-specific R2 transcription as long as the first 17 basepairs of the 5’ untranslated region are present. However, if this 5’ untranslated region is absent, the TATA box is needed for correct initiation of transcription. Focusing on the S phase specificity of the R2 gene expression, we demonstrated that the S phase-specific activity of the mouse R2 promoter is dependent on a protein-binding region located ~500 basepairs upstream of the transcription start site and an E2F binding site close to the transcription start site. Deletion of the upstream activating region results in an inactive promoter. In contrast, mutation of the E2F site leads to premature promoter activation in G1 and increased overall promoter activity. However, if the activating mutation of the E2F site is combined with mutation of the upstream activating region, the promoter becomes inactive. These results suggest that the E2F-dependent regulation is important but not sufficient for cell-cycle specific R2 transcription, and that the upstream activating region is crucial for the overall R2 promoter activity. In our studies of the M phase-specific R2 degradation, we found that it is dependent on a KEN sequence in the N-terminus of the R2 protein, recognized by the Cdh1-APC complex. Mutating the KEN box stabilizes the R2 protein during mitosis and G1 phase. In summary, these studies further extend our understanding of the regulation of the limiting R2 subunit of the enzyme ribonucleotide reductase. The S phase-specific transcription of the R2 gene and the M phase-specific degradation of the R2 protein may serve as important mechanisms to protect the cell against unscheduled DNA synthesis.

Manganese-deficient Streptococcus sanguinis mutants exhibit a dramatic decrease in virulence for infective endocarditis and in aerobic growth in manganese-limited media. Loss of activity of a manganese-dependent, oxygen-dependent ribonucleotide reductase (RNR) could explain the decrease in virulence. When the genes encoding this RNR are deleted, there is no growth of the mutant in aerobic broth culture or in an animal model. Testing the contribution of the aerobic RNR to the phenotype of a manganese transporter mutant, a heterologous class II RNR from Lactobacillus leichmannii called NrdJ that requires B12 rather than manganese as a cofactor was previously introduced into an RNR mutant of S. sanguinis. Aerobic growth was only partially restored. Currently, we sought to improve NrdJ-dependent growth by (i) amending the medium to increase cellular levels of B12; (ii) characterizing a spontaneous mutant of the NrdJ-complemented strain with improved aerobic growth; and (iii) altering this strain through further genetic manipulation.