Site-Specific Self-Catalyzed DNA Depurination: A Biological Mechanism That Leads to Mutations and Creates Sequence Diversity.

Self-catalyzed DNA depurination is a sequence-specific physiological mechanism mediated by spontaneous extrusion of a stem-loop catalytic intermediate. Hydrolysis of the 5'G residue of the 5'GA/TGG loop and of the first 5'A residue of the 5'GAGA loop, together with particular first stem base pairs, specifies their hydrolysis without involving protein, cofactor, or cation. As such, this mechanism is the only known DNA catalytic activity exploited by nature. The consensus sequences for self-depurination of such G- and A-loop residues occur in all genomes examined across the phyla, averaging one site every 2,000-4,000 base pairs. Because apurinic sites are subject to error-prone repair, leading to substitution and short frameshift mutations, they are both a source of genome damage and a means for creating sequence diversity. Their marked overrepresentation in genomes, and largely unchanging density from the lowest to the highest organisms, indicate their selection over the course of evolution. The mutagenicity at such sites in many human genes is associated with loss of function of key proteins responsible for diverse diseases.

Biological role of the major AP (abasic site) endonuclease of an archaeon from geothermal environments.

Archaea and bacteria in geothermal environments are predicted to suffer DNA depurination in vivo at high rates, which raises questions regarding the biological roles of their abasic-site-repair enzymes. Gene deletion and enzymatic assay demonstrated that the saci_0015 gene of Sulfolobus acidocaldarius encodes an AP endonuclease (Apn) accounting for as much as 95% of the assayable activity in cell extracts and is not essential for viability. To identify genetic functions of this enzyme, deletion (ΔApn) strains were examined with respect to growth, spontaneous mutation, transformation by ssDNA containing an abasic site, and conjugation. Relative to its isogenic control, the ΔApn strain did not exhibit any change in growth rate or final cell density, rate or spectrum of spontaneous mutation, transformation by DNA containing an abasic site, or efficiency of DNA transfer and recombination. The apparent lack of genetic impact of removing the major AP endonuclease was unexpected and indicated that abasic sites are rarely bypassed directly by DNA polymerases in S. acidocaldarius. AP endonuclease deficiency had no obvious effect on survival of S. acidocaldarius under several test conditions, but it accelerated the death of cells at 4º C under illumination. Our results suggest that the normal level of AP endonuclease in S. acidocaldarius is well above the minimum required for growth and cell division but not for recovery from prolonged exposure to certain low-temperature conditions. This situation illustrates a biological challenge that has not been emphasized in experimental studies of extremophiles, i.e., the problem of long-term survival under "non-extreme" conditions.

Ribosome depurination by ricin leads to inhibition of endoplasmic reticulum stress-induced HAC1 mRNA splicing on the ribosome.

Ricin undergoes retrograde transport to the endoplasmic reticulum (ER), and ricin toxin A chain (RTA) enters the cytosol from the ER. Previous reports indicated that RTA inhibits activation of the unfolded protein response (UPR) in yeast and in mammalian cells. Both precursor (preRTA) and mature form of RTA (mRTA) inhibited splicing of HAC1u (u for uninduced) mRNA, suggesting that UPR inhibition occurred on the cytosolic face of the ER. Here, we examined the role of ribosome binding and depurination activity on inhibition of the UPR using mRTA mutants. An active-site mutant with very low depurination activity, which bound ribosomes as WT RTA, did not inhibit HAC1u mRNA splicing. A ribosome-binding mutant, which showed reduced binding to ribosomes but retained depurination activity, inhibited HAC1u mRNA splicing. This mutant allowed separation of the UPR inhibition by RTA from cytotoxicity because it reduced the rate of depurination. The ribosome-binding mutant inhibited the UPR without affecting IRE1 oligomerization or cleavage of HAC1u mRNA at the splice site junctions. Inhibition of the UPR correlated with the depurination level, suggesting that ribosomes play a role in splicing of HAC1u mRNA. We show that HAC1u mRNA is associated with ribosomes and does not get processed on depurinated ribosomes, thereby inhibiting the UPR. These results demonstrate that RTA inhibits HAC1u mRNA splicing through its depurination activity on the ribosome without directly affecting IRE1 oligomerization or the splicing reaction and provide evidence that IRE1 recognizes HAC1u mRNA that is associated with ribosomes.

Detection and quantification of ricin-mediated 28S ribosomal depurination by digital droplet PCR.

Ricin acts to damage cells by producing a site of depurination in 28S ribosomal RNA. This depurination results in ribosome inactivation which inhibits protein synthesis and ultimately leads to cell death. We have developed a multiplexed digital droplet polymerase chain reaction assay that enables the objective measurement of toxin activity through quantitation of depurinated 28S rRNA molecules. This assay demonstrates the first use of digital PCR technology to measure ribotoxin-mediated damage. Depurination events were detected in ricin-treated lung cell cultures as early as 1 h, and within 9 h of exposure the maximum ribosomal damage of 70% was reached and was sustained for at least 24 h post-exposure.

Expression of an RNA glycosidase inhibits HIV-1 transactivation of transcription.

HIV-1 (human immunodeficiency virus) transcription is primarily controlled by the virally encoded Tat (transactivator of transcription) protein and its interaction with the viral TAR (transcription response element) RNA element. Specifically, binding of a Tat-containing complex to TAR recruits cellular factors that promote elongation of the host RNA polymerase engaging the viral DNA template. Disruption of this interaction halts viral RNA transcription. In the present study, we investigated the effect of pokeweed antiviral protein (PAP), an RNA glycosidase (EC#: 3.2.2.22) synthesized by the pokeweed plant (Phytolacca americana), on transcription of HIV-1 mRNA. We show that co-expression of PAP with a proviral clone in culture cells resulted in a Tat-dependent decrease in viral mRNA levels. PAP reduced HIV-1 transcriptional activity by inhibiting Tat protein synthesis. The effects of PAP expression on host factors AP-1 (activator protein 1), NF-κB (nuclear factor kappa-light-chain-enhancer of activated B-cells) and specificity protein 1, which modulate HIV-1 transcription by binding to the viral LTR (5'-long terminal repeat), were also investigated. Only AP-1 showed a modest JNK pathway-dependent increase in activity in the presence of PAP; however, this activation was not sufficient to significantly enhance transcription from a partial viral LTR containing AP-1 binding sites. Therefore, the primary effect of PAP on HIV-1 transcription is to reduce viral RNA synthesis by decreasing the abundance of Tat. These findings provide a mechanistic explanation for the observed decrease in viral RNAs in cells expressing PAP and contribute to our understanding of the antiviral effects of this plant protein.

A Role for the Mutagenic DNA Self-Catalyzed Depurination Mechanism in the Evolution of 7SL-Derived RNAs.

The Alu element, the most prevalent SINE (short interspersed element) in the human genome, is one of the many RNA-encoding genes that evolved from the 7SL RNA gene. During analysis of the evolution of 7SL-derived RNAs, two distinct evolutionary intermediates capable of self-catalyzed DNA depurination (SDP) were identified. These SDP sequences spontaneously create apurinic sites that can result in increased mutagenesis due to their error-prone repair. This DNA self-depurination mechanism has been shown both in vitro and in vivo to lead to substitution and short frameshift mutations at a frequency that far exceeds their occurrence due to random errors in DNA replication. In both evolutionary intermediates, the same self-depurination sequence overlaps motifs necessary for successful transcription and SRP9/14 (signal recognition particle) binding; hence, mutations in this region could disrupt RNA activity. Yet, the 7SL-derived RNAs that arose from the elements capable of SDP show significant diversity in this region, and every new sequence retains the transcription and SRP9/14-binding motifs, even as it has lost the SDP sequence. While some (but not all) of the mutagenesis can be alternatively attributed to CpG decay, the very fact that the self-depurinating sequences are selectively discarded in all cases suggests that this was evolutionarily motivated to prevent further destructive mutagenesis by the SDP mechanism.

Toxicity of ricin A chain is reduced in mammalian cells by inhibiting its interaction with the ribosome.

Ricin is a potent ribotoxin that is considered a bioterror threat due to its ease of isolation and possibility of aerosolization. In yeast, mutation of arginine residues away from the active site results in a ricin toxin A chain (RTA) variant that is unable to bind the ribosome and exhibits reduced cytotoxicity. The goal of the present work was to determine if these residues contribute to ribosome binding and cytotoxicity of RTA in mammalian cells. The RTA mutant R193A/R235A did not interact with mammalian ribosomes, while a G212E variant with a point mutation near its active site bound ribosomes similarly to wild-type (WT) RTA. R193A/R235A retained full catalytic activity on naked RNA but had reduced activity on mammalian ribosomes. To determine the effect of this mutant in intact cells, pre R193A/R235A containing a signal sequence directing it to the endoplasmic reticulum and mature R193A/R235A that directly targeted cytosolic ribosomes were each expressed. Depurination and protein synthesis inhibition were reduced by both pre- and mature R193A/R235A relative to WT. Protein synthesis inhibition was reduced to a greater extent by R193A/R235A than by G212E. Pre R193A/R235A caused a greater reduction in caspase activation and loss of mitochondrial membrane potential than G212E relative to WT RTA. These findings indicate that an RTA variant with reduced ribosome binding is less toxic than a variant with less catalytic activity but normal ribosome binding activity. The toxin-ribosome interaction represents a novel target for the development of therapeutics to prevent or treat ricin intoxication.

The molecular characterization of a depurinated trial DNA sample can be a model to understand the reliability of the results in forensic genetics.

The role of DNA damage in PCR processivity/fidelity is a relevant topic in molecular investigation of aged/forensic samples. In order to reproduce one of the most common lesions occurring in postmortem tissues, a new protocol based on aqueous hydrolysis of the DNA was developed in vitro. Twenty-five forensic laboratories were then provided with 3.0 µg of a trial sample (TS) exhibiting, in mean, the loss of 1 base of 20, and a molecular weight below 300 bp. Each participating laboratory could freely choose any combination of methods, leading to the quantification and to the definition of the STR profile of the TS, through the documentation of each step of the analytical approaches selected. The results of the TS quantification by qPCR showed significant differences in the amount of DNA recorded by the participating laboratories using different commercial kits. These data show that only DNA quantification "relative" to the used kit (probe) is possible, being the "absolute" amount of DNA inversely related to the length of the target region (r(2) = 0.891). In addition, our results indicate that the absence of a shared stable and certified reference quantitative standard is also likely involved. STR profiling was carried out selecting five different commercial kits and amplifying the TS for a total number of 212 multiplex PCRs, thus representing an interesting overview of the different analytical protocols used by the participating laboratories. Nine laboratories decided to characterize the TS using a single kit, with a number of amplifications varying from 2 to 12, obtaining only partial STR profiles. Most of the participants determined partial or full profiles using a combination of two or more kits, and a number of amplifications varying from 2 to 27. The performance of each laboratory was described in terms of number of correctly characterized loci, dropped-out markers, unreliable genotypes, and incorrect results. The incidence of unreliable and incorrect genotypes was found to be higher for participants carrying out a limited number of amplifications, insufficient to define the correct genotypes from damaged DNA samples such as the TS. Finally, from a dataset containing about 4500 amplicons, the frequency of PCR artifacts (allele dropout, allele drop-in, and allelic imbalance) was calculated for each kit showing that the new chemistry of the kits is not able to overcome the concern of template-related factors. The results of this collaborative exercise emphasize the advantages of using a standardized degraded DNA sample in the definition of which analytical parameters are critical for the outcome of the STR profiles.

Fragment Screening to Identify Inhibitors Targeting Ribosome Binding of Shiga Toxin 2.

Shiga toxins are the main virulence factors of Shiga toxin producing E. coli (STEC) and S. dysenteriae. There is no effective therapy to counter the disease caused by these toxins. The A1 subunits of Shiga toxins bind the C-termini of ribosomal P-stalk proteins to depurinate the sarcin/ricin loop. The ribosome binding site of Shiga toxin 2 has not been targeted by small molecules. We screened a fragment library against the A1 subunit of Shiga toxin 2 (Stx2A1) and identified a fragment, BTB13086, which bound at the ribosome binding site and mimicked the binding mode of the P-stalk proteins. We synthesized analogs of BTB13086 and identified a series of molecules with similar affinity and inhibitory activity. These are the first compounds that bind at the ribosome binding site of Stx2A1 and inhibit activity. These compounds hold great promise for further inhibitor development against STEC infection.

Programmable deaminase-free base editors for G-to-Y conversion by engineered glycosylase.

Current DNA base editors contain nuclease and DNA deaminase that enables deamination of cytosine (C) or adenine (A), but no method for guanine (G) or thymine (T) editing is available at present. Here we developed a deaminase-free glycosylase-based guanine base editor (gGBE) with G editing ability, by fusing Cas9 nickase with engineered N-methylpurine DNA glycosylase protein (MPG). By several rounds of MPG mutagenesis via unbiased and rational screening using an intron-split EGFP reporter, we demonstrated that gGBE with engineered MPG could increase G editing efficiency by more than 1500 fold. Furthermore, this gGBE exhibited high base editing efficiency (up to 81.2%) and high G-to-T or G-to-C (i.e. G-to-Y) conversion ratio (up to 0.95) in both cultured human cells and mouse embryos. Thus, we have provided a proof-of-concept of a new base editing approach by endowing the engineered DNA glycosylase the capability to selectively excise a new type of substrate.

Mechanism for inhibition of cytotoxicity of Shiga toxin by luteolin.

Enterohemorrhagic or Shiga toxin-producing Escherichia coli is a food-poisoning bacterium that grows in the intestine to produce Shiga toxin (Stx). In this study, the effects of 20 polyphenols on the cytotoxicity of Stx1 and Stx2 in Vero cells were investigated. Among these, epigallocatechin gallate, butein, isorhapontigenin, hesperetin, morin, luteolin, resveratrol, and rhapontigenin showed inhibitory effects on the cytotoxicity of Stxs at 0.4 mmol/L. Furthermore, Vero cells pre-treated with these polyphenols were resistant to Stx at 0.4 mmol/L. However, luteolin showed the most potent inhibitory and cytoprotective effect against Stxs at 0.08 mmol/L or more. This inhibitory mechanism of luteolin was determined using a cell-free protein synthesis system and quantitative reverse transcription PCR assay to detect depurination of 28S rRNA in Vero cells. Luteolin did not inhibit the cell-free protein synthesis by Stxs, suggesting that the enzymatic activity of the Stx A subunit was not inhibited by luteolin. The depurination of 28S rRNA by Stxs was also investigated in Vero cells. The 28S rRNA depurination by Stxs was suppressed in Vero cells treated with Stxs which had been pretreated with luteolin. These results suggest that luteolin inhibits the incorporation of Stxs into Vero cells. This is the first report to show that luteolin inhibits the cytotoxicity of both Stx1 and Stx2 by inhibiting the incorporation of Stxs into Vero cells.

RNA Probes for Visualization of Sarcin/ricin Loop Depurination without Background Fluorescence.

Protein synthesis via ribosomes is a fundamental process in all known living organisms. However, it can be completely stalled by removing a single nucleobase (depurination) at the sarcin/ricin loop of the ribosomal RNA. In this work, we describe the preparation and optimization process of a fluorescent probe that can be used to visualize depurination. Starting from a fluorescent thiophene nucleobase analog, various RNA probes that fluoresce exclusively in the presence of a depurinated sarcin/ricin-loop RNA were designed and characterized. The main challenge in this process was to obtain a high fluorescence signal in the hybridized state with an abasic RNA strand, while keeping the background fluorescence low. With our new RNA probes, the fluorescence intensity and lifetime can be used for efficient monitoring of depurinated RNA.

Chemical Protection and Premodification-Binding Interference for the Identification of Phosphate and Base-Specific Contacts in Protein-DNA Complexes.

The specificity and strength of protein-DNA complexes rely on tight interactions between side- and main chain atoms of amino acid residues and phosphates, sugars, and base-specific groups. Various (in-gel) footprinting methods (for more information, see Chapter 11 ) allow the identification of the global-binding region but do not provide details on the contribution to complex formation of individual sequence-specific constituents of the DNA-binding site. Here, we describe how various chemicals can be used to randomly and sparingly modify specific bases or phosphates and allow the identification of those residues that are specifically protected against modification upon protein binding (protection studies) or interfere with complex formation when modified or removed prior to protein binding (premodification-binding interference). Each one of these complementary approaches has its advantages and shortcomings and results have to be interpreted with caution, having in mind the precise chemistry of the modification. However, used in combination, these methods provide an accurate and high-resolution image of the protein-DNA contacts.

A Simple, Fast and Portable Method for Electrochemical Detection of Adenine Released by Ricin Enzymatic Activity.

International authorities classify ricin toxin present in castor seed as a potential agent for use in bioterrorism. Therefore, the detection, identification, and characterization of ricin in various sample matrices are considered necessary actions for risk assessment during a suspected exposure. This study reports a portable electrochemical assay for detecting active ricin based on the adenine electro-oxidation released from herring sperm DNA substrate by its catalytic action. Also, kinetic parameters were calculated, and the values were Km of 3.14 µM and Kcat 2107 min-1. A linear response was found in optimized experimental conditions for ricin concentrations ranging from 8 to 120 ng/mL, and with a detection limit of 5.14 ng/mL. This proposed detection strategy emphasizes the possibility of field detection of active ricin in food matrices and can be applied to other endonucleolytic activities.

The influence of ricin-mediated rRNA depurination on the translational machinery in vivo - New insight into ricin toxicity.

The generally accepted model of ricin intoxication assumes that direct inactivation of ribosomes by depurination of a specific adenine residue within the sarcin-ricin-loop (SRL) on the 60S ribosomal subunit is a major source of its toxicity. The model proposes that SRL depurination leads to protein synthesis inhibition, evoking ribotoxic stress with concomitant induction of numerous metabolic pathways, which lead to cell death. However, the direct relationship between the depurination and its impact on the translational machinery in vivo has never been satisfactorily explained. In this work, we approached a long-standing question about the influence of SRL depurination on the functioning of the translational machinery in vivo. We have shown that an already low level of depurinated ribosomes exert an effect on cell metabolism, indicating that minute modification within the ribosomal pool is sufficient to elicit a toxic effect. Importantly, depurination does not affect notably any particular step of translation, and translational slowdown caused by ricin is not a direct consequence of depurination and cannot be considered as the sole source of cell death. Instead, SRL depurination in a small fraction of ribosomes blocks cell cycle progression with no effect on cell viability. In this work, we have provided a comprehensive picture of the impact of SRL depurination on the translational apparatus in vivo. We propose that ribosomes with depurinated SRL represent a small imprinted ribosomal pool, which generates a specific signal for the cell to halt the cell cycle.

Inhibition of ricin A-chain (RTA) catalytic activity by a viral genome-linked protein (VPg).

Ricin is a plant derived protein toxin produced by the castor bean plant (Ricinus communis). The Centers for Disease Control (CDC) classifies ricin as a Category B biological agent. Currently, there is neither an effective vaccine that can be used to protect against ricin exposure nor a therapeutic to reverse the effects once exposed. Here we quantitatively characterize interactions between catalytic ricin A-chain (RTA) and a viral genome-linked protein (VPg) from turnip mosaic virus (TuMV). VPg and its N-terminal truncated variant, VPg1-110, bind to RTA and abolish ricin's catalytic depurination of 28S rRNA in vitro and in a cell-free rabbit reticulocyte translational system. RTA and VPg bind in a 1 to 1 stoichiometric ratio, and their binding affinity increases ten-fold as temperature elevates (5 °C to 37 °C). RTA-VPg binary complex formation is enthalpically driven and favored by entropy, resulting in an overall favorable energy, ΔG = -136.8 kJ/mol. Molecular modeling supports our experimental observations and predicts a major contribution of electrostatic interactions, suggesting an allosteric mechanism of downregulation of RTA activity through conformational changes in RTA structure, and/or disruption of binding with the ribosomal stalk. Fluorescence anisotropy studies show that heat affects the rate constant and the activation energy for the RTA-VPg complex, Ea = -62.1 kJ/mol. The thermodynamic and kinetic findings presented here are an initial lead study with promising results and provides a rational approach for synthesis of therapeutic peptides that successfully eliminate toxicity of ricin, and other cytotoxic RIPs.

Polyamines protect nucleic acids against depurination.

Depurination is accelerated by heat and reactive oxygen species under physiological conditions. We previously reported that polyamines are involved in mitigation of heat shock and oxidative stresses through stimulation of the synthesis of heat shock and antioxidant proteins. This time, we investigated whether polyamines are directly involved in protecting nucleic acids from thermal depurination induced by high temperature. The suppressing efficiencies of depurination of DNA by spermine, caldopentamine and caldohexamine in the presence of 1 mM Mg2+, were approximately 50%, 60% and 80%, respectively. Mg2+ also protected nucleic acids against depurination but to a lesser degree than polyamines. Longer unusual polyamines were more effective at protecting DNA against depurination compared to standard polyamines. The tRNA depurination suppressing efficiencies of spermine, caldopentamine and caldohexamine in the presence of 1 mM Mg2+, were approximately 60%, 70% and 80%, respectively. Standard polyamines protected tRNA and ribosomes more effectively than DNA against thermal depurination. Branched polyamines such as mitsubishine and tetrakis(3-aminopropyl)ammonium also protected RNA more effectively than DNA against depurination. These results suggest that the suppressing effect of depurination of nucleic acids (DNA and RNA) depends on the types of polyamines: i.e. to maintain functional conformation of nucleic acids at high temperature, longer and branched polyamines play important roles in protecting nucleic acids from depurination compared to standard polyamines and Mg2+.

Functional Assays for Measuring the Catalytic Activity of Ribosome Inactivating Proteins.

Ribosome-inactivating proteins (RIPs) are potent toxins that inactivate ribosomes by catalytically removing a specific adenine from the α-sarcin/ricin loop (SRL) of the large rRNA. Direct assays for measuring depurination activity and indirect assays for measuring the resulting translation inhibition have been employed to determine the enzyme activity of RIPs. Rapid and sensitive methods to measure the depurination activity of RIPs are critical for assessing their reaction mechanism, enzymatic properties, interaction with ribosomal proteins, ribotoxic stress signaling, in the search for inhibitors and in the detection and diagnosis of enteric infections. Here, we review the major assays developed for measuring the catalytic activity of RIPs, discuss their advantages and disadvantages and explain how they are used in understanding the catalytic mechanism, ribosome specificity, and dynamic enzymatic features of RIPs.

Peptide Mimics of the Ribosomal P Stalk Inhibit the Activity of Ricin A Chain by Preventing Ribosome Binding.

Ricin A chain (RTA) depurinates the sarcin/ricin loop (SRL) by interacting with the C-termini of the ribosomal P stalk. The ribosome interaction site and the active site are located on opposite faces of RTA. The interaction with P proteins allows RTA to depurinate the SRL on the ribosome at physiological pH with an extremely high activity by orienting the active site towards the SRL. Therefore, if an inhibitor disrupts RTA⁻ribosome interaction by binding to the ribosome binding site of RTA, it should inhibit the depurination activity. To test this model, we synthesized peptides mimicking the last 3 to 11 amino acids of P proteins and examined their interaction with wild-type RTA and ribosome binding mutants by Biacore. We measured the inhibitory activity of these peptides on RTA-mediated depurination of yeast and rat liver ribosomes. We found that the peptides interacted with the ribosome binding site of RTA and inhibited depurination activity by disrupting RTA⁻ribosome interactions. The shortest peptide that could interact with RTA and inhibit its activity was four amino acids in length. RTA activity was inhibited by disrupting its interaction with the P stalk without targeting the active site, establishing the ribosome binding site as a new target for inhibitor discovery.

Inhibition of nonenzymatic depurination of nucleic acids by polycations.

DNA base depurination is one of the most common forms of DNA damage in vivo and in vitro, and the suppression of depurination is very important for versatile applications of DNA in biotechnology and medicine. In this work, it was shown that the polycations chitosan (Cho) and spermine (Spm) strongly inhibit DNA depurination through the formation of polyion complexes with DNA molecules. The intramolecular electrostatic interaction of positively charged polycations with DNA efficiently suppresses the protonation of purine groups, which is the key step of depurination. Importantly, the optimal pH for Cho's inhibition of depurination is significantly different from that of Spm. Cho is very effective in the inhibition of depurination in highly acidic media (pH: 1.5-3), whereas Spm is found to suppress the chemical reaction near neutral pH, as well as in acidic solutions. This remarkable pH specificity of the two biorelevant polycations is attributed to the difference in the pKa values of the amino groups. The relevance of our results with the biological roles of biogenic polycations is also discussed.