Formate is the hydrogen donor for the anaerobic ribonucleotide reductase from Escherichia coli.

During anaerobic growth Escherichia coli uses a specific ribonucleoside-triphosphate reductase (class III enzyme) for the production of deoxyribonucleoside triphosphates. In its active form, the enzyme contains an iron-sulfur center and an oxygen-sensitive glycyl radical (Gly-681). The radical is generated in the inactive protein from S-adenosylmethionine by an auxiliary enzyme system present in E. coli. By modification of the previous purification procedure, we now prepared a glycyl radical-containing reductase, active in the absence of the auxiliary reducing enzyme system. This reductase uses formate as hydrogen donor in the reaction. During catalysis, formate is stoichiometrically oxidized to CO2, and isotope from [3H]formate appears in water. Thus E. coli uses completely different hydrogen donors for the reduction of ribonucleotides during anaerobic and aerobic growth. The aerobic class I reductase employs redox-active thiols from thioredoxin or glutaredoxin to this purpose. The present results strengthen speculations that class III enzymes arose early during the evolution of DNA.

Cellular delivery of hammerhead ribozymes

Hammerhead ribozymes are potent RNA molecules which have the potential to specifically inhibit gene expression by catalysing the trans-cleavage of mRNAs. However, they are unstable in biological fluids and cellular delivery poses a problem. Site-specific chemical modification of hammerhead ribozymes was evaluated as a means of enhancing biological stability. Chimeric, 2'-O-methylated ribozymes, containing only five unmodified ribonucleotides, were catalytically active in vitro (kcat = 1.46 min-1) and were significantly more stable in serum and lysosomal enzymes than unmodified (all-RNA) counterparts. Furthermore, they remained undegraded in cell-containing media for up to 8 hours. Stability enhancement allowed cellular uptake properties of radiolabelled ribozymes to be assessed following exogenous delivery. Studies in vulval and glial cell lines indicated that chimeric ribozymes became cell-associated via an inefficient process, which was energy and concentration dependant. A considerable proportion of ribozymes remained bound to cell-surface components, however, a small proportion (<1%) were internalised via mechanisms of adsorptive and / or receptor mediated endocytosis. Fluorescent microscopy indicated that ribozymes were localised within endosomal / lysosomal vesicles following cell entry. This was confirmed by immuno-electron microscopy, which allowed the detection of biotin-labelled ribozymes within the cell ultrastructure. Despite the predominant localisation within endocytic vesicles, a small proportion of internalised ribozymes appeared able to exit these compartments and penetrate target sites within the nucleus and cytoplasm. The ribozymes designed in this report were directed against the epidermal growth factor receptor mRNA, which is over-expressed in a malignant brain disease called glioblastoma multiforme. In order to examine the fate of ribozymes in the brain, the distribution of FITC-labelled ribozymes was examined following intra-cerebro ventricular injection to mice. FITC-ribozymes demonstrated high punctate pattern of distribution within the striatum and cortex, which appeared to represent localisation within cell bodies and dendritic processes. This suggested that delivery to glial cells in vivo may be possible. Finally, strategies were investigated to enhance the cellular delivery of ribozymes. Conjugation of ribozymes to anti~transferrin receptor antibodies improved cellular uptake 3-fold as a result of a specific interaction with transferrin receptors. Complexation with cationic liposomes also significantly improved cell association, however, some toxiclty was observed and this could be a limitation to their use. Overall, it would appear that hammerhead ribozymes can be chemically stabilised to allow direct exogenous administration in vivo. However, additional delivery strategies are probably required to improve cellular uptake, and thus, allow ribozymes to achieve their full potential as pharmaceutical agents. KEYWORDS: Catalytic

Biomimetic modeling of the nitrogen-centered radical postulated to occur during the inhibition of ribonucleotide reductases by 2'-azido-2'-deoxynucleotides

Ribonucleotide reductases (RNR) are essential enzymes that catalyze the reduction of ribonucleotides to 2'-deoxyribonucleotides, which is a critical step that produces precursors for DNA replication and repair. The inactivation of RNR, logically, would discontinue producing the precursors of the DNA of viral or cancer cells, which then would consequently end the cycle of DNA replication. Among different compounds that were found to be inhibitors of RNR, 2'-azido-2'-deoxynucleotide diphosphates (N3NDPs) have been investigated in depth as potent inhibitors of RNR. Decades of investigation has suggested that the inactivation of RNR by N3NDPs is a result of the formation of a nitrogen-centered radical (N·) that is covalently attached to the nucleotide at C3' and cysteine molecule C225 [3'-C(R-S-N·-C-OH)]. Biomimetic simulation reactions for the generation of the nitrogen-centered radicals similar to the one observed during the inactivation of the RNR by azionuclotides was investigated. The study included several modes: (i) theoretical calculation that showed the feasibility of the ring closure reaction between thiyl radicals and azido group; (ii) synthesis of the model azido nucleosides with a linker attached to C3' or C5' having a thiol or vicinal dithiol functionality; (iii) generation of the thiyl radical under both physiological and radiolysis conditions whose role is important in the initiation on RNR cascades; and (iv) analysis of the nitrogen-centered radical species formed during interaction between the thiyl radical and azido group by electron paramagnetic resonance spectroscopy (EPR). Characterization of the aminyl radical species formed during one electron attachment to the azido group of 2'-azido-2'-deoxyuridine and its stereospecifically labelled 1'-, 2'-, 3'-, 4'- or 5,6-[2H 2]-analogues was also examined. This dissertation gave insight toward understanding the mechanism of the formation of the nitrogen-centered radical during the inactivation of RNRs by azidonucleotides as well as the mechanism of action of RNRs that might provide key information necessary for the development of the next generation of antiviral and anticancer drugs.

A model for the modular evolution of rna addressing open questions on the origin of life

A main unsolved problem in the RNA world scenario for the origin of life is how a template-dependent RNA polymerase ribozyme emerged from short RNA oligomers generated by random polymerization of ribonucleotides (Joyce and Orgel 2006). Current estimates establish a minimum size about 165 nt long for such a ribozyme (Johnston et al. 2001), a length three to four times that of the longest RNA oligomers obtained by random polymerization on clay mineral surfaces (Huang and Ferris 2003, 2006). To overcome this gap, we have developed a stepwise model of ligation-based, modular evolution of RNA (Briones et al. 2009) whose main conceptual steps are summarized in Figure 1. This scenario has two main advantages with respect to previous hypotheses put forward for the origin of the RNA world: i) short RNA....

Genetic Analysis of Mitotic Recombination in Saccharomyces cerevisiae

Mitotic genome instability can occur during the repair of double-strand breaks (DSBs) in DNA, which arise from endogenous and exogenous sources. Studying the mechanisms of DNA repair in the budding yeast, Saccharomyces cerevisiae has shown that Homologous Recombination (HR) is a vital repair mechanism for DSBs. HR can result in a crossover event, in which the broken molecule reciprocally exchanges information with a homologous repair template. The current model of double-strand break repair (DSBR) also allows for a tract of information to non-reciprocally transfer from the template molecule to the broken molecule. These “gene conversion” events can vary in size and can occur in conjunction with a crossover event or in isolation. The frequency and size of gene conversions in isolation and gene conversions associated with crossing over has been a source of debate due to the variation in systems used to detect gene conversions and the context in which the gene conversions are measured.

In Chapter 2, I use an unbiased system that measures the frequency and size of gene conversion events, as well as the association of gene conversion events with crossing over between homologs in diploid yeast. We show mitotic gene conversions occur at a rate of 1.3x10-6 per cell division, are either large (median 54.0kb) or small (median 6.4kb), and are associated with crossing over 43% of the time.

DSBs can arise from endogenous cellular processes such as replication and transcription. Two important RNA/DNA hybrids are involved in replication and transcription: R-loops, which form when an RNA transcript base pairs with the DNA template and displaces the non-template DNA strand, and ribonucleotides embedded into DNA (rNMPs), which arise when replicative polymerase errors insert ribonucleotide instead of deoxyribonucleotide triphosphates. RNaseH1 (encoded by RNH1) and RNaseH2 (whose catalytic subunit is encoded by RNH201) both recognize and degrade the RNA in within R-loops while RNaseH2 alone recognizes, nicks, and initiates removal of rNMPs embedded into DNA. Due to their redundant abilities to act on RNA:DNA hybrids, aberrant removal of rNMPs from DNA has been thought to lead to genome instability in an rnh201Δ background.

In Chapter 3, I characterize (1) non-selective genome-wide homologous recombination events and (2) crossing over on chromosome IV in mutants defective in RNaseH1, RNaseH2, or RNaseH1 and RNaseH2. Using a mutant DNA polymerase that incorporates 4-fold fewer rNMPs than wild type, I demonstrate that the primary recombinogenic lesion in the RNaseH2-defective genome is not rNMPs, but rather R-loops. This work suggests different in-vivo roles for RNaseH1 and RNaseH2 in resolving R-loops in yeast and is consistent with R-loops, not rNMPs, being the the likely source of pathology in Aicardi-Goutières Syndrome patients defective in RNaseH2.