Ty1 Retrotransposition Research Articles

Bee venom genotoxicity on Saccharomyces cerevisiae cells – the role of mitochondria and YAP1 transcription factor

The present work aims to clarify the genotype differences of a model organism Saccharomyces cerevisiae in response to bee venom.The study evaluated various endpoints including cell survival, induction of physiologically active superoxide anions, mitotic gene conversion, mitotic crossing-over, reverse mutations, DNA double-strand breaks, and Ty1 retrotransposition. The role of the intact mitochondria and the YAP1 transcription factor was also evaluated.Our results indicate a genotype-specific response. The first experimental evidence has been provided that bee venom induces physiologically active superoxide anions and DNA double-strand breaks in S. cerevisiae. The lack of oxidative phosphorylation due to disrupted or missing mitochondrial DNA reduces but not diminishes the cytotoxicity of bee venom. The possible modes of action could be considered direct damage to membranes (cytotoxic effect) and indirect damage to DNA through oxidative stress (genotoxic effect). YAP1 transcription factor was not found to be directly involved in cell defense against bee venom treatment.

An interchangeable prion-like domain is required for Ty1 retrotransposition

Retrotransposons and retroviruses shape genome evolution and can negatively impact genome function. Saccharomyces cerevisiae and its close relatives harbor several families of LTR-retrotransposons, the most abundant being Ty1 in several laboratory strains. The cytosolic foci that nucleate Ty1 virus-like particle (VLP) assembly are not well understood. These foci, termed retrosomes or T-bodies, contain Ty1 Gag and likely Gag-Pol and the Ty1 mRNA destined for reverse transcription. Here, we report an intrinsically disordered N-terminal prion-like domain (PrLD) within Gag that is required for transposition. This domain contains amino acid composition similar to known yeast prions and is sufficient to nucleate prionogenesis in an established cell-based prion reporter system. Deleting the Ty1 PrLD results in dramatic VLP assembly and retrotransposition defects but does not affect Gag protein level. Ty1 Gag chimeras in which the PrLD is replaced with other sequences, including yeast and mammalian prionogenic domains, display a range of retrotransposition phenotypes from wild type to null. We examine these chimeras throughout the Ty1 replication cycle and find that some support retrosome formation, VLP assembly, and retrotransposition, including the yeast Sup35 prion and the mouse PrP prion. Our interchangeable Ty1 system provides a useful, genetically tractable in vivo platform for studying PrLDs, complete with a suite of robust and sensitive assays. Our work also invites study into the prevalence of PrLDs in additional mobile elements.

A proteomic screen of Ty1 integrase partners identifies the protein kinase CK2 as a regulator of Ty1 retrotransposition

BackgroundTransposable elements are ubiquitous and play a fundamental role in shaping genomes during evolution. Since excessive transposition can be mutagenic, mechanisms exist in the cells to keep these mobile elements under control. Although many cellular factors regulating the mobility of the retrovirus-like transposon Ty1 in Saccharomyces cerevisiae have been identified in genetic screens, only very few of them interact physically with Ty1 integrase (IN).ResultsHere, we perform a proteomic screen to establish Ty1 IN interactome. Among the 265 potential interacting partners, we focus our study on the conserved CK2 kinase. We confirm the interaction between IN and CK2, demonstrate that IN is a substrate of CK2 in vitro and identify the modified residues. We find that Ty1 IN is phosphorylated in vivo and that these modifications are dependent in part on CK2. No significant change in Ty1 retromobility could be observed when we introduce phospho-ablative mutations that prevent IN phosphorylation by CK2 in vitro. However, the absence of CK2 holoenzyme results in a strong stimulation of Ty1 retrotransposition, characterized by an increase in Ty1 mRNA and protein levels and a high accumulation of cDNA.ConclusionOur study shows that Ty1 IN is phosphorylated, as observed for retroviral INs and highlights an important role of CK2 in the regulation of Ty1 retrotransposition. In addition, the proteomic approach enabled the identification of many new Ty1 IN interacting partners, whose potential role in the control of Ty1 mobility will be interesting to study.

Apricot kernels' extract and amygdalin alter bleomycin-induced Ty1 retrotransposition, mitotic gene conversion in the trp-5 locus and reverse point mutations in ilv1-92 allele in Saccharomyces cerevisiae.

The present study aims to analyze the effect of apricot kernels' extract (AKE) and amygdalin (AMY) on bleomycin-induced genetic alternations. Five endpoints were analyzed: cell survival, Ty1 retrotransposition, mitotic gene conversion in the trp-5 locus, reverse point mutations in ilv1-92 allele, and mitotic crossing-over in the ade2 locus. The present work provides the first experimental evidence that bleomycin induces Ty1 retrotransposition in Saccharomyces cerevisiae. New data is obtained that the degree of DNA protection of AMY and AKE depends on the studied genetic event. AKE has been found to provide significant protection against bleomycin-induced Ty1 retrotransposition due to better-expressed antioxidant potential. On the other side, AMY better-expressed protection against bleomycin-induced mitotic gene conversion and reverse mutations may be attributed to the activation of the repair enzymes.

Screening of Amorpha fruticosa and Ailanthus altissima extracts for genotoxicity/antigenotoxicity, mutagenicity/antimutagenicity and carcinogenicity/anticarcinogenicity

The aim of the present study was to evaluate the potential genotoxic/antigenotoxic, mutagenic/antimutagenic, and carcinogenic/anticarcinogenic effect of Amorpha fruticosa (AF) fruit, Ailanthus altissima bark hexane (AAEH) and methanol (AAEM) extracts on a model system Saccharomyces cerevisiae. Plants were identified and extracted by Ekaterina Kozuharova. Three concentrations of each extract were tested – 10, 100 and 1000 µg/ml. In vitro pro-oxidant/antioxidant activities were evaluated by DPPH and DNA topology assay. The potential genotoxic/antigenotoxic, mutagenic/antimutagenic and carcinogenic/anticarcinogenic effects were revealed in vivo by: Zimmermman’s test on Saccharomyces cerevisiae diploid strain D7ts1, and Ty1 retrotransposition test on S. cerevisiae haploid strain 551. Zeocin was used as a positive control. Based on the in vitro antioxidant activity the extracts could be arranged as follows: AF>AAEM>AAEH. AAEH possessed moderate oxidative potential. No genotoxic and mutagenic capacity was obtained in vivo for extracts tested. The levels of total aberrants, convertants and revertants were comparable with the control ones. No Ty1 retrotransposition was induced by extracts treatment. Further, the extracts possessed well-expressed antigenotoxic, antimutagenic and anticarcinogenic activity. Significant reduction of the total aberrants, reverse point mutations and Ty1 retrotransposition was obtained. Only the AF extract was found to reduce the levels of zeocin-induced mitotic gene conversion. The three extracts did not possess any genotoxic, mutagenic and carcinogenic effect on Saccharomyces cerevisiae. Based on their protective activity, they can be arranged as follows: AF>AAEM>AAEH which corresponds well with their phytochemical composition. Further experiments could provide more detailed information concerning the mode of action of extracts, as well as their main constituents.

A nuclear pore sub-complex restricts the propagation of Ty retrotransposons by limiting their transcription.

Beyond their canonical function in nucleocytoplasmic exchanges, nuclear pore complexes (NPCs) regulate the expression of protein-coding genes. Here, we have implemented transcriptomic and molecular methods to specifically address the impact of the NPC on retroelements, which are present in multiple copies in genomes. We report a novel function for the Nup84 complex, a core NPC building block, in specifically restricting the transcription of LTR-retrotransposons in yeast. Nup84 complex-dependent repression impacts both Copia and Gypsy Ty LTR-retrotransposons, all over the S. cerevisiae genome. Mechanistically, the Nup84 complex restricts the transcription of Ty1, the most active yeast retrotransposon, through the tethering of the SUMO-deconjugating enzyme Ulp1 to NPCs. Strikingly, the modest accumulation of Ty1 RNAs caused by Nup84 complex loss-of-function is sufficient to trigger an important increase of Ty1 cDNA levels, resulting in massive Ty1 retrotransposition. Altogether, our study expands our understanding of the complex interactions between retrotransposons and the NPC, and highlights the importance for the cells to keep retrotransposons under tight transcriptional control.

Phytochemical characterization and biological activity of apricot kernels’ extract in yeast-cell based tests and hepatocellular and colorectal carcinoma cell lines

Ethnopharmacological relevanceBitter apricot kernels’ extract contains a broad spectrum of biologically active substances with a lot of attention to amygdalin – cyanogenic glycoside. The extract has been used in the pharmaceutical industry for years as an ingredient of different pharmaceuticals with anti-inflammatory, antimicrobial, or regenerative properties. In traditional medicine, the bitter apricot kernels are known as a remedy for respiratory disorders and skin diseases. The apricot kernels and amygdalin are often prescribed by practitioners for the prevention and treatment of various medical conditions, including colorectal cancer. The present study aimsto evaluate the phytochemical composition and the potential antimutagenic, antirecombinogenic, and antitumor effect of apricot kernels’ extract at very low concentrations in yeast cell-based tests and mammalian hepatocellular and colon carcinoma cell lines. Materials and methodsPhytochemical analysis was performed by LC-MS profiling. Reverse-phase HPLC and UV detection were applied for the determination of amygdalin quantity in the extract. Biological activity was evaluated by Zimmermann's mutagenicity and Ty1 retrotransposition test. Cytotoxic/antiproliferative activity of apricot kernels' extract was performed on four types of cell lines – HepG2, HT-29, BALB/3T3, clone A31, and BJ using the standard MTT-dye reduction assay. ResultsData revealed the presence of more than 1000 compounds and 4 cyanogenic glycosides among them – Amygdalin, Deidaclin, Linamarin and Prulaurasin. The Amygdalin concentration was measured to be 57.8 μg/ml. All extract concentrations demonstrated a strong antigenotoxic, antirecombinogenic, antimutagenic, and anticarcinogenic effect in the yeast cell-based tests. High selectivity of the extract action is established among different mammalian cell lines. Normal cell line BJ is found to be resistant to the extract action. HepG2 was found to be the most sensitive to apricot kernels’ action. ConclusionThe present study provides the first phytochemical analysis of Bulgarian bitter apricot kernels. Three new cyanogenic glycosides were reported. Evidence is obtained that the apricot kernels’ extract at low concentrations is not able to induce some of the events related to the initial steps of tumorigenesis. Additionally, a high selectivity of the extract action is established among different cell lines. The most sensitive cell line was found to be HepG2.

Oxidal® Ameliorates the Ty1 Retrotransposition Induced by Methyl Methanesulfonate in Saccharomyces cerevisiae

Aim: The aim of the study was to evaluate the potential of Oxidal® to decrease the Ty1 retrotransposition rate in a model system Saccharomyces cerevisiae.
 Study Design: Saccharomyces cerevisiae cell suspensions were pre-treated with different concentrations Oxidal® and subsequently treated with 16mM methyl methanesulfonate. (MMS)
 Methodology: The potential of various concentrations Oxidal® was evaluated based on “spot” test and Ty1 retro-transposition test.
 Results: Data revealed that only 5% Oxidal® possesses some cytotoxic properties. Lack of Ty1 retro-transposition was observed after single treatment with 1, 2.5 and 5% Oxidal® concentrations.
 On the other hand, all the tested concentrations showed promising results against the standard carcinogen methyl methane sulfonate. The most pronounced anti-carcinogenic and cytoprotective effects were observed after pre-treatment with 2.5% Oxidal®, which could be attributed to the antioxidant properties of the combination of ingredients; methylene blue, salicylic acid and caffeine. Further studies could reveal the exact mechanism of action of the supplement and the role of the antioxidant potential.
 Conclusion: New data is provided concerning the potential of Oxidal® at low concentrations to protect Saccharomyces cerevisiae cells from MMS-induced Ty1 retro-transposition. The cytoprotective properties of the supplement were also obtained. These results could be considered as a basis for further studies revealing the exact mechanisms of cell protection of the Oxidal®. Additionally, our data could also serve as an important step of the in-depth research of a potential antiviral activity.

Unexplored Molecular Features of the Entamoeba histolytica RNA Lariat Debranching Enzyme Dbr1 Expression Profile

The RNA lariat debranching enzyme (Dbr1) has different functions in RNA metabolism, such as hydrolyzing the 2′-5′ linkage in intron lariats, positively influencing Ty1 and HIV-1 retrotransposition, and modulating snRNP recycling during splicing reactions. It seems that Dbr1 is one of the major players in RNA turnover. It is remarkable that of all the studies carried out to date with Dbr1, to our knowledge, none of them have evaluated the expression profile of the endogenous Dbr1 gene. In this work, we describe, for the first time, that Entamoeba histolytica EhDbr1 mRNA has a very short half-life (less than 30 min) and encodes a very stable protein that is present until trophozoite cultures die. We also show that the EhDbr1 protein is present in the nuclear periphery on the cytoplasmic basal side, contrary to the localization of human Dbr1. Comparing these results with previous hypotheses and with results from different organisms suggests that Dbr1 gene expression is finely tuned and conserved across eukaryotes. Experiments describing the aspects of Dbr1 gene expression and Dbr1 mRNA turnover as well as other functions of the protein need to be performed. Particularly, a special emphasis is needed on the protozoan parasite E. histolytica, the causative agent of amoebiasis, since even though it is a unicellular organism, it is an intron-rich eukaryote whose intron lariats seem to be open to avoid intron lariat accumulation and to process them in non-coding RNAs that might be involved in its virulence.

Binding to RNA regulates Set1 function

The Set1 family of histone H3 lysine 4 (H3K4) methyltransferases is highly conserved from yeast to human. Here we show that the Set1 complex (Set1C) directly binds RNA in vitro through the regions that comprise the double RNA recognition motifs (dRRM) and N-SET domain within Set1 and its subunit Spp1. To investigate the functional relevance of RNA binding, we performed UV RNA crosslinking (CRAC) for Set1 and RNA polymerase II in parallel with ChIP-seq experiments. Set1 binds nascent transcripts through its dRRM. RNA binding is important to define the appropriate topology of Set1C distribution along transcription units and correlates with the efficient deposition of the H3K4me3 mark. In addition, we uncovered that Set1 binds to different classes of RNAs to levels that largely exceed the levels of binding to the general population of transcripts, suggesting the Set1 persists on these RNAs after transcription. This class includes RNAs derived from SET1, Ty1 retrotransposons, specific transcription factors genes and snRNAs (small nuclear RNAs). We propose that Set1 modulates adaptive responses, as exemplified by the post-transcriptional inhibition of Ty1 retrotransposition.

Antimutagenic, Antirecombinogenic, and Antitumor Effect of Amygdalin in a Yeast Cell-Based Test and Mammalian Cell Lines.

Amygdalin is a major component of the seeds of Rosaceae family of plants such as apricots, peaches, cherry, nectarines, apples, plums, and so on, as well as almonds. It is used in alternative medicine for cancer prevention, alleviation of fever, cough suppression, and quenching thirst. The aim of the present study is to determine the mutagenic and recombinogenic effects of amygdalin in a test system Saccharomyces cerevisiae and to evaluate its potential antitumor effect in a yeast cell-based test and colon cancer cell lines. Results obtained show that concentrations 25, 50, and 100 μg/mL did not have any cytotoxic, mutagenic, and carcinogenic effect in yeast cell-based tests. Pretreatment with amygdalin at concentration 100 μg/mL leads to around twofold of the cell survival and decrease of reverse mutation frequency, induced by the alkylating agent methyl methanesulfonate. The frequency of gene conversion and mitotic crossing-over is around threefold lower. The anticarcinogenic potential of amygdalin at the same concentration is presented as around fourfold reduction of Ty1 retrotransposition induced by hexavalent chromium. In summary, data presented in this study provide evidence concerning the inability of amygdalin itself to provoke events related to the initial steps of tumorigenesis. In addition, the observed antimutagenic/antirecombinogenic effect could be activation of error-free and error-prone recombination events. Based on the high selectivity toward normal or tumor cell lines, it could be speculated that amygdalin has higher cytotoxic effect in cell lines with higher proliferative and metabolic activity, which are the majority of fast developing tumors.

Ty1 escapes restriction by the self-encoded factor p22 through mutations in capsid

Ty1 is a long terminal repeat (LTR) retrotransposon belonging to the Ty1/copia family and is present in up to 32 full-length copies in Saccharomyces. Like retroviruses, Ty1 contains GAG and POL genes, LTRs, and replicates via an RNA intermediate within a virus-like particle (VLP). Although Ty1 retrotransposition is not infectious, uncontrolled replication can lead to detrimental effects on the host genome, including insertional mutagenesis and chromosomal rearrangements. Ty1 copy number control (CNC) limits replication and is mediated through a self-encoded protein called p22. p22 is translated from a subgenomic Ty1 RNA and encodes an amino-truncated version of the Gag protein. We highlight a recent study identifying Ty1 Gag, which comprises the VLP capsid and provides nucleic acid chaperone functions, as a direct target of p22-mediated inhibition. CNC-resistant (CNCR) mutations map within predicted helical domains of Gag, including those in the Ty1/copia pfam domain Retrotran_gag_2 (formerly UBN2) and a central region we refer to as the CNCR domain. CNCR Gag forms VLPs that exclude p22, thus restoring Ty1 replication. We discuss possible mechanisms for p22 inclusion in Ty1 VLPs and compare Ty1 CNC with retroviral restriction factors targeting capsid (CA).

Characterizing the functions of Ty1 Gag and the Gag-derived restriction factor p22/p18.

The long terminal repeat (LTR) and non-LTR retrotransposons comprise approximately half of the human genome, and we are only beginning to understand their influence on genome function and evolution. The LTR retrotransposon Ty1 is the most abundant mobile genetic element in the S. cerevisiae reference genome. Ty1 replicates via an RNA intermediate and shares several important structural and functional characteristics with retroviruses. However, unlike retroviruses Ty1 retrotransposition is not infectious. Retrotransposons integrations can cause mutations and genome instability. Despite the fact that S. cerevisiae lacks eukaryotic defense mechanisms such as RNAi, they maintain a relatively low copy number of the Ty1 retrotransposon in their genomes. A novel restriction factor derived from the C-terminal half of Gag (p22/p18) and encoded by internally initiated transcript inhibits retrotransposition in a dose-dependent manner. Therefore, Ty1 evolved a specific GAG organization and expression strategy to produce products both essential and antagonistic for retrotransposon movement. In this commentary we discuss our recent research aimed at defining steps of Ty1 replication influenced by p22/p18 with particular emphasis on the nucleic acid chaperone functions carried out by Gag and the restriction factor.

A self-encoded capsid derivative restricts Ty1 retrotransposition in Saccharomyces.

Retrotransposons and retroviral insertions have molded the genomes of many eukaryotes. Since retroelements transpose via an RNA intermediate, the additive nature of the replication cycle can result in massive increases in copy number if left unchecked. Host organisms have countered with several defense systems, including domestication of retroelement genes that now act as restriction factors to minimize propagation. We discovered a novel truncated form of the Saccharomyces Ty1 retrotransposon capsid protein, dubbed p22 that inhibits virus-like particle (VLP) assembly and function. The p22 restriction factor expands the repertoire of defense proteins targeting the capsid and highlights a novel host-parasite strategy. Instead of inhibiting all transposition by domesticating the restriction gene as a distinct locus, Ty1 and budding yeast may have coevolved a relationship that allows high levels of transposition when Ty1 copy numbers are low and progressively less transposition as copy numbers rise. Here, we offer a perspective on p22 restriction, including its mode of expression, effect on VLP functions, interactions with its target, properties as a nucleic acid chaperone, similarities to other restriction factors, and future directions.

The Ty1 Retrotransposon Restriction Factor p22 Targets Gag.

A novel form of copy number control (CNC) helps maintain a low number of Ty1 retrovirus-like transposons in the Saccharomyces genome. Ty1 produces an alternative transcript that encodes p22, a trans-dominant negative inhibitor of Ty1 retrotransposition whose sequence is identical to the C-terminal half of Gag. The level of p22 increases with copy number and inhibits normal Ty1 virus-like particle (VLP) assembly and maturation through interactions with full length Gag. A forward genetic screen for CNC-resistant (CNCR) mutations in Ty1 identified missense mutations in GAG that restore retrotransposition in the presence of p22. Some of these mutations map within a predicted UBN2 domain found throughout the Ty1/copia family of long terminal repeat retrotransposons, and others cluster within a central region of Gag that is referred to as the CNCR domain. We generated multiple alignments of yeast Ty1-like Gag proteins and found that some Gag proteins, including those of the related Ty2 elements, contain non-Ty1 residues at multiple CNCR sites. Interestingly, the Ty2-917 element is resistant to p22 and does not undergo a Ty1-like form of CNC. Substitutions conferring CNCR map within predicted helices in Ty1 Gag that overlap with conserved sequence in Ty1/copia, suggesting that p22 disturbs a central function of the capsid during VLP assembly. When hydrophobic residues within predicted helices in Gag are mutated, Gag level remains unaffected in most cases yet VLP assembly and maturation is abnormal. Gag CNCR mutations do not alter binding to p22 as determined by co-immunoprecipitation analyses, but instead, exclude p22 from Ty1 VLPs. These findings suggest that the CNCR alleles enhance retrotransposition in the presence of p22 by allowing productive Gag-Gag interactions during VLP assembly. Our work also expands the strategies used by retroviruses for developing resistance to Gag-like restriction factors to now include retrotransposons.

RTT105 in Ty1 Retrotransposition: a Possible Role in Virus‐like Particle (VLP) Assembly

Retrovirus‐like transposon mRNA encodes envelope and replication proteins (gag and pol), and serves as genomic material for virus‐like particles (VLPs), a cytosolic structure in which the element's genome is reverse transcribed. In Saccharomyces cerevisiae, there are several families of such retrotransposons, including the abundant Ty1 element. Previously, genome‐wide screens have identified candidate genes that mediate Ty1 mobility. One candidate, RTT105, was identified as a negative regulator of Ty1 mobility. Because RTT105 contains no conserved domains, the nature of RTT105's role in Ty1 retrotransposition remains unknown. Rtt105p localizes at Ty1 RNA and Gag foci, suggesting a role in ribonucleoprotein complexes. Here, we use RNA‐Seq analysis of isogenic WT and RTT105 knockout strains to determine changes in gene expression that may explain RTT105's regulation of Ty1. In rtt105Δ, we identified significant down regulation of signal recognition particle (SRP), a conserved chaperone that binds ribosome‐nascent chain complexes for post‐transcriptional processing. SRP aids in translocating Gag to the ER where it encapsulates Ty1 RNA in trans at presumptive VLP assembly sites. Because endogenous Ty1 mobility is maintained at low levels by the host cell, our data suggest that RTT105 may support SRP‐dependent processing of Ty1. Deletion of RTT105 may induce down regulation of SRP‐dependent processing of Ty1, thereby decreasing genome‐wide mobility of the element. This result illuminates an important step in retrotransposon processing and regulation, and suggests at least one route by which Ty1 activity is actively maintained by the cell.This research was funded by HHMI, NIH, and the Huck Institutes of the Life Sciences.

A trans-dominant form of Gag restricts Ty1 retrotransposition and mediates copy number control.

Saccharomyces cerevisiae and Saccharomyces paradoxus lack the conserved RNA interference pathway and utilize a novel form of copy number control (CNC) to inhibit Ty1 retrotransposition. Although noncoding transcripts have been implicated in CNC, here we present evidence that a truncated form of the Gag capsid protein (p22) or its processed form (p18) is necessary and sufficient for CNC and likely encoded by Ty1 internal transcripts. Coexpression of p22/p18 and Ty1 decreases mobility more than 30,000-fold. p22/p18 cofractionates with Ty1 virus-like particles (VLPs) and affects VLP yield, protein composition, and morphology. Although p22/p18 and Gag colocalize in the cytoplasm, p22/p18 disrupts sites used for VLP assembly. Glutathione S-transferase (GST) affinity pulldowns also suggest that p18 and Gag interact. Therefore, this intrinsic Gag-like restriction factor confers CNC by interfering with VLP assembly and function and expands the strategies used to limit retroelement propagation. Retrotransposons dominate the chromosomal landscape in many eukaryotes, can cause mutations by insertion or genome rearrangement, and are evolutionarily related to retroviruses such as HIV. Thus, understanding factors that limit transposition and retroviral replication is fundamentally important. The present work describes a retrotransposon-encoded restriction protein derived from the capsid gene of the yeast Ty1 element that disrupts virus-like particle assembly in a dose-dependent manner. This form of copy number control acts as a molecular rheostat, allowing high levels of retrotransposition when few Ty1 elements are present and inhibiting transposition as copy number increases. Thus, yeast and Ty1 have coevolved a form of copy number control that is beneficial to both "host and parasite." To our knowledge, this is the first Gag-like retrotransposon restriction factor described in the literature and expands the ways in which restriction proteins modulate retroelement replication.

Comprehensive Mechanistic Analysis of the RNA‐Lariat Debranching Enzyme

RNA‐lariat Debranching Enzyme (Dbr1p) hydrolyzes the 2′‐5′ phosphodiester bond between the intronic, adenosine branch point and the 5′ end of the intron. Intron lariat debranching is essential for processing of snoRNAs and miRNAs and for efficient intron turnover. Interestingly, Dbr1p null mutants have been shown to be defective in Ty1 retrotransposition in Saccharomyces cerevisiae. Additionally, siRNA knockdown of DBR1 in a eukaryotic cell line has inhibited HIV‐1 replication, making characterization and structure determination imperative to the rational design of a wide range of inhibitors.We present structural, mechanistic and comparative analyses of two Dbr1 proteins. While S. cerevisiae Dbr1p has been characterized biochemically, complete mechanistic understanding has been limited by the unavailability of physiologically relevant substrates, as well as the lack of a high‐resolution Dbr1p structure. Our comprehensive analysis has utilized in vitro cleavage assays with a variety of branched substrates, site‐directed mutagenesis, inductively coupled plasma atomic emission spectroscopy (ICP‐AES) and fluorescence anisotropy to characterize Dbr1p kinetics, and to identify a unique versatility in substrate recognition and a hetero‐dinuclear active site.Funding is provided by the CMU Center for Nucleic Acids Science and Technology, NSF GRFP #0750271 and the UNCF/Merck Graduate Fellowship.

Retrotransposition is associated with genome instability during chronological aging

Genetic damage through mutations and genome rearrangements has been hypothesized to contribute to aging. The specific mechanisms responsible for age-induced increases in mutation and chromosome rearrangement frequencies and a potential causative role for DNA damage in aging are under active investigation. Retrotransposons are mobile genetic elements that cause insertion mutations and contribute to genome rearrangements through nonallelic recombination events in humans and other organisms. We have investigated the role of endogenous Ty1 retrotransposons in aging-associated increases in genome instability using the Saccharomyces cerevisiae chronological aging model. We show that age-induced increases in loss of heterozygosity and chromosome loss events are consistently diminished by mutations or treatments that reduce Ty1 retrotransposition. Ty1 mobility is elevated in very old yeast populations, and new retromobility events are often associated with chromosome rearrangements. These results reveal a correlation between retrotransposition and genome instability during yeast aging. Retrotransposition may contribute to genetic damage during aging in diverse organisms and provides a useful tool for studying whether genetic damage is a causative factor for aging.

Characterization of mutant alleles of RTT105 in Saccharomyces cerevisiae

Saccharomyces cerevisiae, commonly known as baker's yeast, contains several retrotransposons within its genome, including the Ty1 element. There are many host genes that have been shown to regulate Ty1 retrotransposition, including RTT105; however, the host function of this gene is unknown. RTT105 was originally identified as a negative Ty1 regulator by the insertion of the bacterial transposon, Tn3, creating a disruption at the 212bp of the 627bp gene. The resulting mutant allele, rtt105::Tn3‐LacZ/Leu2, increases transposition of an endogenous Ty1 element. However, our lab isolated the RTT105 gene in a screen of genes that when over‐expressed, increased retrotransposition of a galactose inducible Ty1 element on a 2‐micron (high copy) plasmid. We have shown that a complete deletion of RTT105 results in dramatically less galactose‐induced Ty1 retrotransposition. From this, we hypothesize that the Tn3 allele does not nullify gene function but rather enhances it. Real‐time quantitative PCR and northern blots are being used to determine expression levels of the wild type and rtt105::Tn3‐LacZ/Leu2 allele from RNA extracts of the strains. Since the Tn3 insert is over 7kbp in size, disruption mutants at the same 212bp location are being made with auxotrophic markers varying in length. This will help determine whether expression of the N‐terminal region of RTT105 specifically inhibits transposition or whether sequences within Tn3 may be impacting RTT105 expression. The various insertion mutants will be assayed for retrotransposition and RNA extracts will be used to look at expression levels of the mutant rtt105 alleles. Additionally, we are purifying the Rtt105 protein for antibody production by expressing in E. coli. This research is supported by NIH grant GM074658‐01 to JBK.