Sequence-specific binding of Taz1p dimers to fission yeast telomeric DNA

The fission yeast (Schizosaccharomyces pombe) taz1 gene encodes a telomere-associated protein. It contains a single copy of a Myb-like motif termed the telobox that is also found in the human telomere binding proteins TRF1 and TRF2, and Tbf1p, a protein that binds to sequences found within the sub-telomeric regions of budding yeast (Saccharomyces cerevisiae) chromosomes. Taz1p was synthesised in vitro and shown to bind to a fission yeast telomeric DNA fragment in a sequence specific manner that required the telobox motif. Like the mammalian TRF proteins, Taz1p bound to DNA as a preformed homodimer. The isolated Myb-like domain was also capable of sequence specific DNA binding, although with less specificity than the full-length dimer. Surprisingly, a protein extract produced from a taz1–fission yeast strain still contained the major telomere binding activity (complex I) we have characterised previously, suggesting that there could be other abundant telomere binding proteins in fission yeast. One candidate, SpX, was also synthesised in vitro, but despite the presence of two telobox domains, no sequence specific binding to telomeric DNA was detected.     

Evolution of Telomeres in Schizosaccharomyces pombe and Its Possible Relationship to the Diversification of Telomere Binding Proteins

Telomeres of nuclear chromosomes are usually composed of an array of tandemly repeated sequences that are recognized by specific Myb domain containing DNA-binding proteins (telomere-binding proteins, TBPs). Whereas in many eukaryotes the length and sequence of the telomeric repeat is relatively conserved, telomeric sequences in various yeasts are highly variable. Schizosaccharomyces pombe provides an excellent model for investigation of co-evolution of telomeres and TBPs. First, telomeric repeats of S. pombe differ from the canonical mammalian type TTAGGG sequence. Second, S. pombe telomeres exhibit a high degree of intratelomeric heterogeneity. Third, S. pombe contains all types of known TBPs (Rap1p [a version unable to bind DNA], Tay1p/Teb1p, and Taz1p) that are employed by various yeast species to protect their telomeres. With the aim of reconstructing evolutionary paths leading to a separation of roles between Teb1p and Taz1p, we performed a comparative analysis of the DNA-binding properties of both proteins using combined qualitative and quantitative biochemical approaches. Visualization of DNA-protein complexes by electron microscopy revealed qualitative differences of binding of Teb1p and Taz1p to mammalian type and fission yeast telomeres. Fluorescence anisotropy analysis quantified the binding affinity of Teb1p and Taz1p to three different DNA substrates. Additionally, we carried out electrophoretic mobility shift assays using mammalian type telomeres and native substrates (telomeric repeats, histone-box sequences) as well as their mutated versions. We observed relative DNA sequence binding flexibility of Taz1p and higher binding stringency of Teb1p when both proteins were compared directly to each other. These properties may have driven replacement of Teb1p by Taz1p as the TBP in fission yeast.     

The telomere protein Taz1 is required to prevent and repair genomic DNA breaks

One fundamental function of telomeres is to prevent the ends of chromosomes from being sensed and treated as DNA damage. Here we present evidence for additional roles of telomeres in promoting proper chromosome segregation and DNA repair. We find that the fission yeast telomere protein Taz1p is required for cell cycle progression at 20 degrees C, a temperature at which taz1Delta cells exhibit a G(2)/M DNA damage checkpoint delay, chromosome missegregation, and DNA double-strand breaks (DSBs). Spindle assembly checkpoint components and a checkpoint-independent function of Rad3p are required for taz1Delta cells to survive at 20 degrees C. Disruption of topoisomerase II activity suppresses the cold sensitivity of taz1Delta cells, suggesting a scenario in which telomeric entanglement is the primary defect. Furthermore, hypersensitivity to treatments that induce DSBs suggests that Taz1p is involved in DSB repair. Our observations imply roles for Taz1p-containing telomeres in preventing and repairing DNA breaks throughout the genome.     

Recombination-based telomere maintenance is dependent on Tel1-MRN and Rap1 and inhibited by telomerase, Taz1, and Ku in fission yeast

Fission yeast cells survive loss of the telomerase catalytic subunit Trt1 (TERT) through recombination-based telomere maintenance or through chromosome circularization. Although trt1Δ survivors with linear chromosomes can be obtained, they often spontaneously circularize their chromosomes. Therefore, it was difficult to establish genetic requirements for telomerase-independent telomere maintenance. In contrast, when the telomere-binding protein Taz1 is also deleted, taz1Δ trt1Δ cells are able to stably maintain telomeres. Thus, taz1Δ trt1Δ cells can serve as a valuable tool in understanding the regulation of telomerase-independent telomere maintenance. In this study, we show that the checkpoint kinase Tel1 (ATM) and the DNA repair complex Rad32-Rad50-Nbs1 (MRN) are required for telomere maintenance in taz1Δ trt1Δ cells. Surprisingly, Rap1 is also essential for telomere maintenance in taz1Δ trt1Δ cells, even though recruitment of Rap1 to telomeres depends on Taz1. Expression of catalytically inactive Trt1 can efficiently inhibit recombination-based telomere maintenance, but the inhibition requires both Est1 and Ku70. While Est1 is essential for recruitment of Trt1 to telomeres, Ku70 is dispensable. Thus, we conclude that Taz1, TERT-Est1, and Ku70-Ku80 prevent telomere recombination, whereas MRN-Tel1 and Rap1 promote recombination-based telomere maintenance. Evolutionarily conserved proteins in higher eukaryotic cells might similarly contribute to telomere recombination.     

The fission yeast MRN complex tethers dysfunctional telomeres for NHEJ repair

Telomeres protect the natural ends of chromosomes from being repaired as deleterious DNA breaks. In fission yeast, absence of Taz1 (homologue of human TRF1 and TRF2) renders telomeres vulnerable to DNA repair. During the G1 phase, when non‐homologous end joining (NHEJ) is upregulated, taz1Δ cells undergo telomere fusions with consequent loss of viability. Here, we show that disruption of the fission yeast MRN (Rad23^MRE11‐Rad50‐Nbs1) complex prevents NHEJ at telomeres and, as a result, rescues taz1Δ lethality in G1. Neither Tel1^ATM activation nor 5′‐end resection was required for telomere fusion. Nuclease activity of Rad32^MRE11 was also dispensable for NHEJ. Mutants unable to coordinate metal ions required for nuclease activity were proficient in NHEJ repair. In contrast, Rad32^MRE11 mutations that affect binding and/or positioning of DNA ends leaving the nuclease function largely unaffected also impaired NHEJ at telomeres and restored the viability of taz1Δ in G1. Consistently, MRN structural integrity but not nuclease function is also required for NHEJ of independent DNA ends in a novel split‐molecule plasmid assay. Thus, MRN acts to tether unlinked DNA ends, allowing for efficient NHEJ.     

The cardiolipin transacylase, tafazzin, associates with two distinct respiratory components providing insight into Barth syndrome

Mutations in the mitochondrial cardiolipin (CL) transacylase, tafazzin (Taz1p), result in the X-linked cardioskeletal myopathy, Barth syndrome (BTHS). The mitochondria of BTHS patients exhibit variable respiratory defects and abnormal cristae ultrastructure. The biochemical basis for these observations is unknown. In the absence of its target phospholipid, CL, a very large Taz1p complex is missing, whereas several discrete smaller complexes are still observed. None of the identified Taz1p complexes represents Taz1p homodimers. Instead, yeast Taz1p physically assembles in several protein complexes of distinct size and composition. The ATP synthase and AAC2, both required for oxidative phosphorylation, are identified in separate stable Taz1p complexes. In the absence of CL, each interaction is still detected albeit in reduced abundance compared with when CL is present. Taz1p is not necessary for the normal expression of AAC2 or ATP synthase subunits or assembly of their respective complexes. In contrast, the largest Taz1p complex requires assembled ATP synthase and CL. Mitochondria in Δtaz1 yeast, similar to ATP synthase oligomer mutants, exhibit altered cristae morphology even though ATP synthase oligomer formation is unaffected. Thus, the Taz1p interactome defined here provides novel insight into the variable respiratory defects and morphological abnormalities observed in mitochondria of BTHS patients.     

A non‐canonical function of topoisomerase II in disentangling dysfunctional telomeres

The decatenation activity of topoisomerase II (Top2), which is widely conserved within the eukaryotic domain, is essential for chromosomal segregation in mitosis. It is less clear, however, whether Top2 performs the same function uniformly across the whole genome, and whether all its functions rely on decatenation. In the fission yeast, Schizosaccharomyces pombe, telomeres are bound by Taz1, which promotes smooth replication fork progression through the repetitive telomeric sequences. Hence, replication forks stall at taz1Δ telomeres. This leads to telomeric entanglements at low temperatures (⩽20°C) that cause chromosomal segregation defects and loss of viability. Here, we show that the appearance of entanglements, and the resulting cold sensitivity of taz1Δ cells, is suppressed by mutated alleles of Top2 that confer slower catalytic turnover. This suppression does not rely on the decatenation activity of Top2. Rather, the enhanced presence of reaction intermediates in which Top2 is clamped around DNA, promotes the removal of telomeric entanglements in vivo, independently of catalytic cycle completion. We propose a model for how the clamped enzyme–DNA complex promotes proper chromosomal segregation.     

Efficient expression of truncated recombinant cadmium-metallothionein gene of a ciliate, Tetrahymena tropicalis lahorensis in Escherichia coli

Truncated recombinant metallothionein GST–fusion protein has been successfully expressed in Escherichia coli. The previously identified novel Cd-inducible metallothionein (TMCd1) gene from the locally isolated ciliate, Tetrahymena tropicalis lahorensis, was inserted into a pET-41a vector, in frame with a sequence encoding an N-terminal glutathione-S-transferase (GST) tail. Truncated recombinant GST fusion protein has been purified by affinity column chromatography using glutathione sepharose. After enzymatic cleavage of GST tail with enterokinase, the truncated TMCd1 MT shows molecular weight of 11.5 kDa, corresponding to the expected value. This is the first successful report of expression of cadmium metallothionein gene of a ciliate, T. t. lahorensis, reported from this part of the world, in E. coli. This study will further help in characterization of metallothionein protein of this ciliate.     

The N-Terminal Region of the RecU Holliday Junction Resolvase Is Essential for Homologous Recombination

The RecU Holliday junction (HJ)-resolving enzyme is highly conserved in the Firmicutes phylum of bacteria. In Bacillus subtilis, the recU gene has two putative initiation codons, at positions 1 and 33. In rec⁺ cells, only the full-length RecU polypeptide (206 residues, 23.9 kDa) was detected even after different stress treatments. To address the relevance of the flexible N-terminus, we constructed mutant variants. Experiments in vivo revealed that recUΔ1-32 (which initiates at Met33 and encodes RecUΔ1-32) and recU31 (the conserved Arg31 residue was substituted with alanine to give RecUR31A) are genuine RecU mutants, rendering cells impaired in DNA repair and chromosomal segregation. RecU has three activities: It (i) cleaves HJs, (ii) anneals complementary strands and (iii) modulates RecA activities. RecUR31A binds and cleaves HJ DNA in vitro as efficiently as wild-type RecU, but RuvB·ATPγS·Mg²⁺ fails to stimulate the RecUR31A cleavage reaction. In contrast, RecUΔ1-32 forms unstable complexes with DNA and fails to cleave HJs. RecU and its variants are capable of promoting DNA strand annealing and exert a negative effect on deoxy-ATP-dependent RecA-mediated DNA strand exchange. This study shows that the flexible N-terminus of RecU is essential for protein activity.     

Expression, purification, and immunogenic characterization of Epstein–Barr virus recombinant EBNA1 protein in Pichia pastoris

Epstein–Barr virus (EBV) is a ubiquitous human herpesvirus associated with the development of both lymphoid and epithelial tumors. EBNA1 is the only viral protein expressed in all EBV-associated malignancies and plays important roles in EBV latency. Thus, EBNA1 is thought to be a promising antigen for immunotherapy of all EBV-associated malignancies. This study was undertaken to produce recombinant EBNA1 protein in Pichia pastoris and evaluate its immunogenicity. The truncated EBNA1 (E1ΔGA, codons 390–641) was expressed as a secretory protein with an N-terminal histidine tag in the methylotrophic yeast P. pastoris and purified by Ni-NTA affinity chromatography. The purified proteins were then used as antigens to immunize BALB/c mice for production of polyclonal antibodies. Western blot analysis showed that the polyclonal antibodies specifically recognized the EBNA1 protein in B95-8 cell lysates. The recombinant E1ΔGA also induced strong lymphoproliferative and Th1 cytokine responses in mice. Furthermore, mice immunized with E1ΔGA developed CD4⁺ and CD8⁺ T cell responses. These findings showed that the yeast-expressed E1ΔGA retained good immunogenicity and might be a promising vaccine candidate against EBV-associated malignancies.     

Structural transition from dimeric to tetrameric i‐motif,caused by the presence of TAA at the 3′‐end of human telomeric C‐rich sequence

Widely dispersed in genomic DNA, the tandem C‐rich repetitive stretches may fold below physiological pH, into i‐motif structures, stabilized by C·C⁺ pairing. Herein, structural status of a 9‐mer stretch d(CCCTAACCC), [the truncated double repeat of human telomeric sequence], and its extended version, comprising of additional ? TAA segment at the 3′‐end, representing the complete double repeat d(CCCTAACCCTAA), has been investigated. The pH dependent monophasic UV‐melting, Gel and CD data suggested that while the truncated version adopts a bimolecular i‐motif structure, its complete double repeat (12‐mer) sequence exists in two (bimolecular and tetramolecular) forms. A model is proposed for the tetramolecular i‐motif with conventional C · C⁺ base pairs, additionally stabilized by asymmetric A · A base pairs at the ?3′ TAA flanking ends and Watson–Crick A · T hydrogen bonding between intervening bases on antiparallel strands. Expanding the known topologies of DNA i‐motifs, such atypical geometries of i‐motifs may have implications in their recognition by proteins. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 150–160, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Fission yeast Rap1 homolog is a telomere-specific silencing factor and interacts with Taz1p

Taz1p is the fission yeast orthologue of human TRF2, a telomeric repeat-binding protein. Delta(taz1) mutants are defective in telomeric silencing, telomere length control, and meiotic recombination events. A recent report demonstrated that the human Rap1p homolog (hRap1) is recruited to telomere by interaction with TRF2, arguing that the telomere control mechanism of higher eukaryotes is distinct from that of the budding yeast. Taz1p showed a significant similarity to human TRF2, but not with the budding yeast Rap1p (scRap1p). This suggests that Taz1p and TRF2 share common features in telomere regulation. To assess the roles of Taz1p in telomere-related functions in detail, we attempted to identify a protein(s) that interacts with Taz1p by using two-hybrid screening. Interestingly, the sequence analysis of a positive clone revealed a perfect match with a Rap1 homolog in S. pombe (spRap1), which showed a significant homology with scRap1p and hRap1p. Here we show that the spRap1 deficiency in haploid cells is viable, which results in increased telomere length regulation, disruption of telomere silencing, and aberrant meiosis (like the delta(taz1) mutant). This suggests that spRap1p might be recruited to the telomere by Taz1p and play crucial roles in telomere function. Interestingly, the delta(rap1) mutants in fission yeast are defective only for telomere silencing. Therefore, the role of spRap1p may be distinct from that of scRap1p, which is involved in the silencing at both the telomere and mating type locus. Our data, therefore, suggest that the regulation mechanisms of telomere in fission yeast resemble that of higher eukaryotic cells rather than the budding yeast.     

The fission yeast Taz1 protein protects chromosomes from Ku-dependent end-to-end fusions

A paramount role of telomeres is to prevent chromosome fusions. The fission yeast Taz1 protein regulates diverse telomere functions but is not essential for growth under stress-free conditions. Strikingly, however, taz1(-) cells exhibit lethal telomere fusions when subjected to nitrogen starvation, a treatment that induces an uncommitted G1 state. These fusions are formed by Ku-dependent nonhomologous end joining. Fusions also occur during normal growth in taz1(-) cells that lack rad22(+), a gene involved in homologous recombination. Our data suggest a model whereby taz1(-) telomeres are exposed to the prevailing mode of DNA repair, which is dictated by the cell cycle. Thus, Taz1 caps chromosome ends and provides the telomerespecific interaction that prevents Ku from treating telomeres as double-strand breaks.     

Cloning and truncation modification of trehalose-6-phosphate synthase gene from Selaginella pulvinata

A homologous sequence was amplified from resurrection plant Selaginella pulvinta by RACE technique, proved to be the full-length cDNA of trehalose-6-phosphate synthase gene by homologous alignment and yeast complementation assay, and nominated as SpTPS1 gene. The open reading frame of this gene was truncated 225 bp at the 5′-end, resulting the N-terminal truncation modification of 75 amino acids for its encoding protein. The TPS1 deletion mutant strain YSH290 of the brewer's yeast transformed by the truncated gene SpTPS1Δ and its original full-length version restored growth on the medium with glucose as a sole carbon source and displayed growth curves with no significant difference, indicating their encoding proteins functioning as TPS enzyme. The TPS activity of the mutant strain transformed by the truncated gene SpTPS1Δ was about six fold higher than that transformed by its original version, reasoning that the extra N-terminal extension of the full-length amino acid sequence acts as an inhibitory domain to trehalose synthesis. However, the trehalose accumulation of the mutant strain transformed by the truncated gene SpTPS1Δ was only 8% higher than that transformed by its original version. This result is explained by the feedback balance of trehalose content coordinated by the comparative activities between trehalose synthase and trehalase. The truncated gene SpTPS1Δ is suggested to be used in transgenic operation, together with the inhibition of trehalase activity by the application of validamycin A or genetic deficiency of the endogenous trehalase gene, for the enhancement of trehalose accumulation and improvement of abiotic tolerance in transgenic plants.     

In vitro methylation of bacteriophage lambda DNA by wild type (dam+) and mutant (damh) forms of the phage T2 DNA adenine methylase.

The wild-type (dam⁺) and mutant (dam^h) forms of the bacteriophage T2 DNA adenine methylase have been partially purified; these enzymes methylate the sequence, 5/t' … G-A-Py … 3′ (Hattman et al., 1978a). However, in vitro methylation studies using phage λ DNA revealed the following: (1) T2 dam⁺ and dam^h enzymes differ in their ability to methylate λ DNA; under identical reaction conditions the T2 dam^h enzyme methylated λ DNA to a higher level than did the dam⁺ enzyme. However, the respective methylation sites are equally distributed on the l and r strands. (2) Methylation with T2 dam^h, but not T2 dam⁺ protected λ against P1 restriction. This was demonstrated by transfection of Escherichia coli (P1) spheroplasts and by cleavage with R·EcoP1. (3) T2 dam⁺ and dam^h were similarly capable of methylating G-A-T-C sequences on λ DNA; e.g. λ·dam₃ DNA (contains no N⁶-methyladonine) methylated with either enzyme was made resistant to cleavage by R·DpnII. In contrast, only the T2 dam^h modified DNA was resistant to further methylation by M·EcoP1 (which methylates the sequence 5′ … A-G-A-C-Py … 3′; Hattman et al., 1978b). (4) λ·dam₃ DNA was partially methylated to the same level with T2 dam⁺ or T2 dam^h; the two enzymes produced different patterns of G-A-C versus G-A-T methylation. We propose that the T2 dam⁺ enzyme methylates G-A-C sequences less efficiently than the T2 dam^h methylase; this property does not entirely account for the large difference in methylation levels produced by the two enzymes.     

Self-association of isolated large cytoplasmic domain of plasma membrane H-ATPase from Saccharomyces cerevisiae: Role of the phosphorylation domain in a general dimeric model for P-ATPases

Large cytoplasmic domain (LCD) plasma membrane H⁺-ATPase from S. cerevisiae was expressed as two fusion polypeptides in E. coli: a DNA sequence coding for Leu353-Ileu674 (LCDh), comprising both nucleotide (N) and phosphorylation (P) domains, and a DNA sequence coding for Leu353-Thr543 (LCDΔh, lacking the C-terminus of P domain), were inserted in expression vectors pDEST-17, yielding the respective recombinant plasmids. Overexpressed fusion polypeptides were solubilized with 6 M urea and purified on affinity columns, and urea was removed by dialysis. Their predicted secondary structure contents were confirmed by CD spectra. In addition, both recombinant polypeptides exhibited high-affinity 2′,3′-O-(2,4,6-trinitrophenyl)adenosine-5′-triphosphate (TNP-ATP) binding (K_d = 1.9 μM and 2.9 μM for LCDh and LCDΔh, respectively), suggesting that they have native-like folding. The gel filtration profile (HPLC) of purified LCDh showed two main peaks, with molecular weights of 95 kDa and 39 kDa, compatible with dimeric and monomeric forms, respectively. However, a single elution peak was observed for purified LCDΔh, with an estimated molecular weight of 29 kDa, as expected for a monomer. Together, these data suggest that LCDh exist in monomer-dimer equilibrium, and that the C-terminus of P domain is necessary for self-association. We propose that such association is due to interaction between vicinal P domains, which may be of functional relevance for H⁺-ATPase in native membranes. We discuss a general dimeric model for P-ATPases with interacting P domains, based on published crystallography and cryo-electron microscopy evidence.     

The conversion of L-lysine to saccharopine and alpha-aminoadipate in mouse

Saccharopine [?-N-(l-glutaryl-2)-l-lysine] has been found to occur in normal, untreated mouse liver. The pool of saccharopine as well as that of α-aminoadipate become labeled shortly after the administration of l-lysine-U-¹⁴C into intact mouse. In vitro experiments using the mouse liver homogenate have shown that l-lysine is converted to saccharopine in the presence of α-ketoglutarate and NADPH, and saccharopine to α-aminoadipate in the presence of NAD⁺. The oxidation of α-aminoadipic-δ-semialdehyde (Δ¹-piperideine-6-carboxylate), the proposed reaction product of saccharopine cleavage, to α-aminoadipate is effected by either NAD⁺ or NADP⁺.     

The armadillo repeat domain of Apc suppresses intestinal tumorigenesis

The adenomatous polyposis coli (APC) gene is known to act as a tumor suppressor gene in both sporadic and hereditary colorectal cancer by negatively regulating WNT signaling. Familial adenomatous polyposis (FAP) patients develop intestinal polyps due to the presence of a single germline mutation in APC. The severity of the FAP phenotype is a function of the position of the APC mutation, indicating a complex role for APC that extends beyond the canonical WNT pathway. APC encodes a large protein with multiple functional domains, including an armadillo repeat domain that has been linked to protein–protein interactions. To determine the effect of the armadillo repeat domain on intestinal tumorigenesis, we generated a congenic mouse line (Apc ^Δ242 ) carrying a gene trap cassette between exons 7 and 8 of the murine Apc gene. Apc ^Δ242/+ mice express a truncated Apc product lacking the armadillo repeat domain as part of a fusion protein with β-geo. Expression of the fusion product was confirmed by X-gal staining, ensuring that Apc ^Δ242 is not a null allele. In contrast, Apc ^Min/+ mice produce a truncated Apc product that contains an intact armadillo repeat domain. On the C57BL/6J background, Apc ^Δ242/+ mice develop more polyps than do Apc ^Min/+ mice along the entire length of the small intestine; however, polyps were significantly smaller in Apc ^Δ242/+ mice. In addition, polyp multiplicity in Apc ^Δ242/+ mice is affected by polymorphisms between inbred strains. These data suggest that the armadillo repeat domain of the Apc protein suppresses tumor initiation in the murine intestine while also promoting tumor growth.