The Recombinases Rad51 and Dmc1 Play Distinct Roles in DNA Break Repair and Recombination Partner Choice in the Meiosis of Tetrahymena

Repair of programmed DNA double-strand breaks (DSBs) by meiotic recombination relies on the generation of flanking 3′ single-stranded DNA overhangs and their interaction with a homologous double-stranded DNA template. In various common model organisms, the ubiquitous strand exchange protein Rad51 and its meiosis-specific homologue Dmc1 have been implicated in the joint promotion of DNA–strand exchange at meiotic recombination sites. However, the division of labor between these two recombinases is still a puzzle. Using RNAi and gene-disruption experiments, we have studied their roles in meiotic recombination and chromosome pairing in the ciliated protist Tetrahymena as an evolutionarily distant meiotic model. Cytological and electrophoresis-based assays for DSBs revealed that, without Rad51p, DSBs were not repaired. However, in the absence of Dmc1p, efficient Rad51p-dependent repair took place, but crossing over was suppressed. Immunostaining and protein tagging demonstrated that only Dmc1p formed strong DSB–dependent foci on meiotic chromatin, whereas the distribution of Rad51p was diffuse within nuclei. This suggests that meiotic nucleoprotein filaments consist primarily of Dmc1p. Moreover, a proximity ligation assay confirmed that little if any Rad51p forms mixed nucleoprotein filaments with Dmc1p. Dmc1p focus formation was independent of the presence of Rad51p. The absence of Dmc1p did not result in compensatory assembly of Rad51p repair foci, and even artificial DNA damage by UV failed to induce Rad51p foci in meiotic nuclei, while it did so in somatic nuclei within one and the same cell. The observed interhomologue repair deficit in dmc1Δ meiosis is consistent with a requirement for Dmc1p in promoting the homologue as the preferred recombination partner. We propose that relatively short and/or transient Rad51p nucleoprotein filaments are sufficient for intrachromosomal recombination, whereas long nucleoprotein filaments consisting primarily of Dmc1p are required for interhomolog recombination

Tetrahymena Metallothioneins Fall into Two Discrete Subfamilies

BACKGROUND: Metallothioneins are ubiquitous small, cysteine-rich, multifunctional proteins which can bind heavy metals. METHODOLOGY/PRINCIPAL FINDINGS: We report the results of phylogenetic and gene expression analyses that include two new Tetrahymena thermophila metallothionein genes (MTT3 and MTT5). Sequence alignments of all known Tetrahymena metallothioneins have allowed us to rationalize the structure of these proteins. We now formally subdivide the known metallothioneins from the ciliate genus Tetrahymena into two well defined subfamilies, 7a and 7b, based on phylogenetic analysis, on the pattern of clustering of Cys residues, and on the pattern of inducibility by the heavy metals Cd and Cu. Sequence alignment also reveals a remarkably regular, conserved and hierarchical modular structure of all five subfamily 7a MTs, which include MTT3 and MTT5. The former has three modules, while the latter has only two. Induction levels of the three T. thermophila genes were determined using quantitative real time RT-PCR. Various stressors (including heavy metals) brought about dramatically different fold-inductions for each gene; MTT5 showed the highest fold-induction. Conserved DNA motifs with potential regulatory significance were identified, in an unbiased way, upstream of the start codons of subfamily 7a MTs. EST evidence for alternative splicing in the 3′ UTR of the MTT5 mRNA with potential regulatory activity is reported. CONCLUSION/SIGNIFICANCE: The small number and remarkably regular structure of Tetrahymena MTs, coupled with the experimental tractability of this model organism for studies of in vivo function, make it an attractive system for the experimental dissection of the roles, structure/function relationships, regulation of gene expression, and adaptive evolution of these proteins, as well as for the development of biotechnological applications for the environmental monitoring of toxic substances

Autophagy and lipid droplets are a defense mechanism against toxic copper oxide nanotubes in the eukaryotic microbial model Tetrahymena thermophila

The widespread use of inorganic nanomaterials of anthropogenic origin has significantly increased in the last decade, being now considered as emerging pollutants. This makes it necessary to carry out studies to further understand their toxicity and interactions with cells. In the present work we analyzed the toxicity of CuO nanotubes (CuONT) in the ciliate Tetrahymena thermophila, a eukaryotic unicellular model with animal biology. CuONT exposure rapidly induced ROS generation in the cell leading to oxidative stress and upregulation of genes encoding antioxidant enzymes (catalase, superoxide dismutase, glutathione peroxidase), metal-chelating metallothioneins and cytochrome P450 monooxygenases. Comet assays and overexpression of genes involved in DNA repair confirmed oxidative DNA damage in CuONT-treated cells. Remarkably, both electron and fluorescent microscopy revealed numerous lipid droplets and autophagosomes containing CuONT aggregates and damaged mitochondria, indicating activation of macroautophagy, which was further confirmed by a dramatic upregulation of ATG (AuTophaGy related) genes. Treatment with autophagy inhibitors significantly increased CuONT toxicity, evidencing the protective role of autophagy towards CuONTinduced damage. Moreover, increased formation of lipid droplets appears as an additional mechanism of CuONT detoxification. Based on these results, we present a hypothetical scenario summarizing how T. thermophila responds to CuONT toxicity. This study corroborates the use of this ciliate as an excellent eukaryotic microbial model for analyzing the cellular response to stress caused by toxic metal nanoparticles

Roles of the Tetrahymena thermophila type I element binding factor, TIF1, in DNA replication and genome stability

The Tetrahymena thermophila rDNA minichromosome has been used as a model system for studying DNA replication. Previous studies have identified cis-acting replication determinants within the rDNA origin and promoter region including the type I element that is essential for replication initiation, fork progression and promoter activation. TIF1 is a non-ORC single strand-binding protein that binds the type I element in vivo. TIF1 binds opposing strands at the origin and promoter regions indicating that it may play a role in selectively marking these regions. In this dissertation, I use gene disruption to elucidate the role of TIF1 in replication. This work reveals that TIF1 represses rDNA origin firing, and is required for proper macronuclear S phase progression and division. Replication at the rDNA origin initiates precociously despite the observation that TIF1 mutants exhibit an elongated macronuclear S phase and a diminished rate of DNA replication. The amitotic macronucleus also displays delayed and abnormal division even though cells exit S phase with a wild-type macronuclear DNA content. Nuclear defects are also evident in the diploid micronucleus as TIF1 mutants contain fewer micronuclear chromosomes and are unable to pass genetic information to progeny. This defect is progressive as clonal mutant lines exhibit micronuclear instability during subsequent vegetative cell cycling. This work reveals that these macro- and micronuclear phenotypes may be the result of DNA damage as TIF1 mutants are hypersensitive to DNA damaging agents. This suggests that TIF1 mutants may have defects in the DNA damage response pathway. TIF1-deficient cells also incur DNA damage with no exogenous damaging agents. I propose that micro- and macronuclear defects witnessed in TIF1 mutant cells result from cells exiting S phase with compromised chromosomes due to the accumulation of DNA damage. Furthermore, TIF1 appears to play a role in the prevention, recognition or repair of DNA damage in addition to regulating rDNA replication and cell cycle progression and division. Additionally, TIF1 plays an essential role in the faithful propagation of both the macro- and micronuclear genomes

Genome-wide identification and characterization of cytochrome P450 monooxygenase genes in the ciliate Tetrahymena thermophila

<p>Abstract</p> <p>Background</p> <p>Cytochrome P450 monooxygenases play key roles in the metabolism of a wide variety of substrates and they are closely associated with endocellular physiological processes or detoxification metabolism under environmental exposure. To date, however, none has been systematically characterized in the phylum Ciliophora. <it>T. thermophila </it>possess many advantages as a eukaryotic model organism and it exhibits rapid and sensitive responses to xenobiotics, making it an ideal model system to study the evolutionary and functional diversity of the P450 monooxygenase gene family.</p> <p>Results</p> <p>A total of 44 putative functional cytochrome P450 genes were identified and could be classified into 13 families and 21 sub-families according to standard nomenclature. The characteristics of both the conserved intron-exon organization and scaffold localization of tandem repeats within each P450 family clade suggested that the enlargement of <it>T. thermophila </it>P450 families probably resulted from recent separate small duplication events. Gene expression patterns of all <it>T. thermophila </it>P450s during three important cell physiological stages (vegetative growth, starvation and conjugation) were analyzed based on EST and microarray data, and three main categories of expression patterns were postulated. Evolutionary analysis including codon usage preference, site-specific selection and gene-expression evolution patterns were investigated and the results indicated remarkable divergences among the <it>T. thermophila </it>P450 genes.</p> <p>Conclusion</p> <p>The characterization, expression and evolutionary analysis of <it>T. thermophila </it>P450 monooxygenase genes in the current study provides useful information for understanding the characteristics and diversities of the P450 genes in the Ciliophora, and provides the baseline for functional analyses of individual P450 isoforms in this model ciliate species.</p

Conserved and Unconventional Responses to DNA Damage in Tetrahymena

Here the ciliate protozoa Tetrahymena thermophila was used as a model system to study the DNA damage response. Tetrahymena enclose nuclear dimorphism, a polyploid somatic macronucleus (MAC), which is transcriptionally active and maintains vegetative growth, and a diploid germline micronucleus (MIC) responsible for the transmition of genetic information during conjugation. Previous studies have identified Tif1p, a novel protein involved in the regulation of rDNA replication in Tetrahymena. TIF1 hypomorphic strains acquire spontaneous DNA damage during vegetative cell cycle and are hypersensitive to DNA damaging agents. TIF1-deficient strains acquire DNA damage in both nuclear compartments, suggesting a global role of Tif1p in the maintenance of genomic stability. In my dissertation research, I studied the role of Tif1p during the cell cycle progression. To this end, I generated tagged-Tif1p strains, which revealed that the subcellular localization of Tif1p is dynamic throughout the cell cycle. However, the addition of epitope tag to this protein generated phenotypes analogous to ones observed in a TIF1-deficient strain. This suggested that the addition of epitope tag to Tif1p severely affects the properties of Tif1p and hence the overall integrity of the cell. To overcome these limitations, a peptide antibody specific to Tif1p was generated to study the endogenous protein. This work revealed that the abundance of Tif1p protein is not cell cycle regulated and that Tif1p is absent in starved cells. Furthermore, the specific binding of TIf1p to rDNA minichromosome was studied during vegetative cell cycles. Chromatin immunoprecipitation studies revealed that the specific binding of Tif1p extends beyond the cis-acting determinant of replication present at the rDNA origin and promoter. This suggests that coding regions may be targeted for the binding of Tif1p to previously uncharacterized sequences, and that Tif1p preferentially localizes on the rDNA minichromosome. I also studied the induction of DNA damage response, demonstrating that Tetrahymena activates a checkpoint response mediated by an ATR-like pathway. Studies with a hypomorphic TIF1 strain revealed that Tif1p mediates proper activation of the DNA damage response. Further characterization of the response to genotoxic agents showed that Tetrahymena is able to activate a G1/S and intra-S phase DNA damage response. The results presented here suggest that a caffeine-dependent checkpoint activator protein modulates the response to DNA damage. In addition, a subunit of the replicative helicase, Mcm6p, is directly affected by the induction of DNA damage. This suggests that Tetrahymena uses a novel mechanism to halt the progression of DNA replication forks during genotoxic stress through degradation of Mcm6p

Characterization of Histone H2A Functional Domains Important for Regulation of the DNA Damage Response

DNA double strand breaks represent deleterious lesions which can either be caused by environmental or endogenous sources of DNA damage. Efficient DNA damage response which ensures repair of these lesions is therefore critical for maintenance of genomic stability. The repair happens in the context of chromatin, a three-dimensional nucleoprotein complex consisting of DNA, histones and associated proteins. As such, mechanisms that modulate chromatin structure, many of which involve the histone component of chromatin, have been shown to play a role in regulation of the DNA damage response. In my thesis work I characterize two conserved histone H2A functional domains that are required for normal response to DNA damage. In the first part of my thesis, my collaborators and I demonstrate that Tetrahymena major histone H2A.S contains an H2A.X variant-specific SQ motif within its C-terminal tail, providing the first description of this region in ciliated protozoa. The function of the SQ motif is mediated by post-translational phosphorylation of the conserved serine which is essential for normal progression through Tetrahymena life cycle, and in particular, meiosis. This study provides the first evidence for the existence of meiotic DSBs in Tetrahymena and defines the time interval of meiotic recombination in this organism. In the second part of my thesis, I describe a functional domain which encodes a unique and previously unrecognized role for the histone H2A Nterminal tail in the DNA damage response in S. cerevisiae. A DNA damage survival property exists within the conserved SRS motif spanning residues 17-19 of a single turn α-helical region in the H2A tail, known as the ‘knuckle’. I demonstrate that the SRS motif is required for efficient checkpoint recovery following successful repair, a function independent of post-translational modifications. Another contribution of histone H2A in S. cerevisiae, specific to the MMS induced DNA damage response, is provided by the three amino-terminal lysines which appear to be functionally redundant. My collaborators and I demonstrate that in vivo two of the lysines, H2A K4 and H2A K7, are acetylated individually as well as together , and identify the third lysine, H2A K13, as a novel acetylation site in S. cerevisiae

Effects of altered expression of the sumo conjugating enzyme, UBC9 on mitosis, meiosis and conjugation in Tetrahymena thermophila

SUMOylation is a critical posttranslational modification in eukaryotic species. Ubc9p is the E2-conjugating enzyme for SUMOylation and consequently it influences multiple cellular pathways. Nuclear proteins are common targets of SUMOylation and regulate nuclear events such as transcription, DNA repair and mitosis. The segregation of the Tetrahymena thermophila genome into two different nuclear compartments provides an unusual context for the analysis of SUMOylation. Each cell contains a transcriptionally silent, diploid germ line micronucleus (MIC) that divides by mitosis and a polyploid transcriptionally active somatic macronucleus (MAC) that divides by an amitotic mechanism. With the long-term goal to exploit these opportunities we initiated studies of Ubc9p and therefore indirectly SUMOylation, on the functionally distinct nuclei in T. thermophila using genetic analysis combined with proteomics study. We found that complete deletion of the UBC9 gene is lethal. Rescue of the lethal phenotype with a GFP-UBC9 fusion gene driven by a metallothionein promoter generated a cell line with a slow growth phenotype in the absence of CdCl 2-dependent expression of GFP-Ubc9p. Altered expression of Ubc9p resulted in differential effects in MICs and MACs. MICs were lost from cells during vegetative growth but MACs were capable of division. Interestingly, cells expressing a catalytically inactive dominant negative Ubc9p (DN-Ubc9p) accumulated multiple MICs. Ubc9p depleted cells were hypersensitive to DNA damaging agents that promote double-strand DNA breaks. Additional studies point to critical roles for Ubc9p during the sexual life cycle of Tetrahymena. Crosses between cell lines expressing the dominant negative Ubc9p were delayed in meiosis and produced fewer exconjugant progeny who successfully completed genetic exchange and conjugation than from wild-type controls. In contrast, cell lines that were depleted for Ubc9p did not form pairs and therefore could not complete any of the subsequent stages of conjugation including meiosis and macronuclear development. The results are consistent with roles for Ubc9p in mitosis, meiosis and double strand break repair. A proteomics-based approach generated an unbiased spectrum of Ubc9p interacting proteins during Tetrahymena vegetative growth and conjugation. We identified 128 high-confidence Ubc9p interacting proteins duringTetrahymena vegetative growth and 106 proteins during conjugation, among which 58 are conjugation-specific. Seven proteins with homologs in other species have been reported previously as SUMO substrates, or Ubc9p interacting proteins. The Ubc9p interactome covers a wide range of cellular processes, including chromatin remodeling, cell cycle progression, stress response, gene transcription and Tetrahymena macronuclear development, which further support our observations from phenotypic analysis. The findings provide evidence for distinct roles for SUMOylation in ciliate nuclei and provide opportunities for future studies of SUMOylated substrates in a context specific for gene expression (MAC) or mitotic and meiotic division (MIC)

Evolution of Telomerase RNA

abstract: The highly specialized telomerase ribonucleoprotein enzyme is composed minimally of telomerase reverse transcriptase (TERT) and telomerase RNA (TR) for catalytic activity. Telomerase is an RNA-dependent DNA polymerase that syntheizes DNA repeats at chromosome ends to maintain genome stability. While TERT is highly conserved among various groups of species, the TR subunit exhibits remarkable divergence in primary sequence, length, secondary structure and biogenesis, making TR identification extremely challenging even among closely related groups of organisms. A unique computational approach combined with in vitro telomerase activity reconstitution studies was used to identify 83 novel TRs from 10 animal kingdom phyla spanning 18 diverse classes from the most basal sponges to the late evolving vertebrates. This revealed that three structural domains, pseudoknot, a distal stem-loop moiety and box H/ACA, are conserved within TRs from basal groups to vertebrates, while group-specific elements emerge or disappear during animal TR evolution along different lineages. Next the corn-smut fungus Ustilago maydis TR was identified using an RNA-immunoprecipitation and next-generation sequencing approach followed by computational identification of TRs from 19 additional class Ustilaginomycetes fungi, leveraging conserved gene synteny among TR genes. Phylogenetic comparative analysis, in vitro telomerase activity and TR mutagenesis studies reveal a secondary structure of TRs from higher fungi, which is also conserved with vertebrates and filamentous fungi, providing a crucial link in TR evolution within the opisthokonta super-kingdom. Lastly, work by collabarotors from Texas A&M university and others identified the first bona fide TR from the model plant Arabidopsis thaliana. Computational analysis was performed to identify 85 novel AtTR orthologs from three major plant clades: angiosperms, gymnosperms and lycophytes, which facilitated phylogenetic comparative analysis to infer the first plant TR secondary structural model. This model was confirmed using site-specific mutagenesis and telomerase activity assays of in vitro reconstituted enzyme. The structures of plant TRs are conserved across land plants providing an evolutionary bridge that unites the disparate structures of previously characterized TRs from ciliates and vertebrates.Dissertation/ThesisDoctoral Dissertation Biochemistry 201

Analysis of DIE5 and LIA5 reveals the importance of DNA repair in programmed DNA rearrangement of Tetrahymena thermophila

During its somatic nuclear differentiation, the single cell eukaryote Tetrahymena thermophila undergoes genome-wide programmed DNA rearrangement to eliminate transposon-like elements from its future soma. This process involves small RNA-directed heterochromatin formation followed by extensive nuclear reorganization to form subnuclear domains. While more has been known about small RNAs and heterochromatin, the mechanisms and players involved in the process of nuclear reorganization and the subsequent removal of transposon-like elements from the somatic genome are just starting to unravel. My thesis work centers on the study of two novel nuclear proteins Die5p: Chapter 2) and Lia5p: Chapter 3) and their roles in DNA rearrangement. These essential proteins function downstream of small RNA targeted heterochromatin establishment. While Lia5p is essential for nuclear reorganization to form distinct subnuclear structures, Die5p is a protein conserved across ciliate species and appears to be important for the integrity of the differentiating genome. Maintaining genome integrity during somatic nuclear differentiation has proven to be an active process. Similar to V(D)J recombination during mammalian B and T cell maturation, programmed DNA rearrangement in Tetrahymena induces global DNA damage that requires proper response and repair. Through the study of LIA5 and DIE5, we show that nuclear reorganization during Tetrahymena DNA rearrangement is intimately associated with the response to DNA damage. Furthermore, we implicate a chromodomain protein Pdd1 as a component of the DNA damage response system, thus providing evidence to support the link between heterochromatin and DNA repair during the reprogramming of Tetrahymena somatic genome