Def1p is involved in telomere maintenance in budding yeast

Saccharomyces Rrm3p, a member of Pif1 5'-3' DNA helicase subfamily, helps replication forks traverse protein-DNA complexes, including the telomere. Here we have identified an Rrm3p interaction protein known to be Def1p. In def1 mutants, telomeres were approximately 200-bp shorter than that in wild-type cells. DEF1 is also required for the stable maintenance of mitochondrial DNA, and the telomere shortening phenotype seen in def1 cells is not a secondary consequence of the mitochondrion defect. A combination of DEF1 null mutation with deletion of EST2 or EST3 resulted in an accelerated senescence phenotype, suggesting that Def1p is not involved in the telomerase recruitment pathway. In the absence of telomerase, cells escape senescence by either amplifying Y' regions or TG-telomeric repeats to generate type I or type II survivors, respectively. Only type I survivors were recovered from both def1Delta est2Delta and def1Delta est3Delta double mutant cells, further suggesting that the function of Def1p in telomere maintenance is specific. Our novel findings of the functions of Def1p in telomere and mitochondria suggested that Def1p plays multiple roles in yeast.     

Telomerase dependence of telomere lengthening in Ku80 mutant Arabidopsis

We have identified a ku80 mutant of Arabidopsis and show that telomerase is needed to generate the longer telomeres observed in this mutant. Telomeres are specialized nucleoprotein structures at the ends of chromosomes that permit cells to distinguish chromosome ends from double-strand breaks, thus preventing chromosome fusion events. Ku80 deficiency results in the lengthening of telomeres, a phenotype also seen in an Arabidopsis ku70 mutant. Furthermore, homogeneous populations of ku80 mutant cells show a steady increase in the length of telomere tracts, which reach an equilibrium length and then stabilize. In contrast to that in mammals, Ku80 deficiency in Arabidopsis cells does not cause end-to-end fusion of chromosomes. This telomere lengthening is dependent on the presence of telomerase, although it is not attributable to a significant increase in telomerase activity per se. These results demonstrate the essential role of the Ku80 protein as a negative regulator of telomerase function in plant cells.     

Eme1 is involved in DNA damage processing and maintenance of genomic stability in mammalian cells

Yeast and human Eme1 protein, in complex with Mus81, constitute an endonuclease that cleaves branched DNA structures, especially those arising during stalled DNA replication. We identified mouse Eme1, and show that it interacts with Mus81 to form a complex that preferentially cleaves 3'-flap structures and replication forks rather than Holliday junctions in vitro. We demonstrate that Eme1-/- embryonic stem (ES) cells are hypersensitive to the DNA cross-linking agents mitomycin C and cisplatin, but only mildly sensitive to ionizing radiation, UV radiation and hydroxyurea treatment. Mammalian Eme1 is not required for the resolution of DNA intermediates that arise during homologous recombination processes such as gene targeting, gene conversion and sister chromatid exchange (SCE). Unlike Blm-deficient ES cells, increased SCE was seen only following induced DNA damage in Eme1-deficient cells. Most importantly, Eme1 deficiency led to spontaneous genomic instability. These results reveal that mammalian Eme1 plays a key role in DNA repair and the maintenance of genome integrity.     

The effect of Ku on telomere replication time is mediated by telomere length but is independent of histone tail acetylation

DNA replication in Saccharomyces cerevisiae proceeds according to a temporal program. We have investigated the role of the telomere-binding Ku complex in specifying late replication of telomere-proximal sequences. Genome-wide analysis shows that regions extending up to 80 kb from telomeres replicate abnormally early in a yku70 mutant. We find that Ku does not appear to regulate replication time by binding replication origins directly, nor is its effect on telomere replication timing mediated by histone tail acetylation. We show that Ku instead regulates replication timing through its effect on telomere length, because deletion of the telomerase regulator Pif1 largely reverses the short telomere defect of a yku70 mutant and simultaneously rescues its replication timing defect. Consistent with this conclusion, deleting the genome integrity component Elg1 partially rescued both length and replication timing of yku70 telomeres. Telomere length-mediated control of replication timing requires the TG(1-3) repeat-counting component Rif1, because a rif1 mutant replicates telomeric regions early, despite having extended TG(1-3) tracts. Overall, our results suggest that the effect of Ku on telomere replication timing results from its impact on TG(1-3) repeat length and support a model in which Rif1 measures telomere repeat length to ensure that telomere replication timing is correctly programmed.     

ATM is required for telomere maintenance and chromosome stability during Drosophila development

ATM is a large, multifunctional protein kinase that regulates responses required for surviving DNA damage: including DNA repair, apoptosis, and cell cycle checkpoints. Here, we show that Drosophila ATM function is essential for normal adult development. Extensive, inappropriate apoptosis occurs in proliferating atm mutant tissues, and in clonally derived atm mutant embryos, frequent mitotic defects were seen. At a cellular level, spontaneous telomere fusions and other chromosomal abnormalities are common in atm larval neuroblasts, suggesting a conserved and essential role for dATM in the maintenance of normal telomeres and chromosome stability. Evidence from other systems supports the idea that DNA double-strand break (DSB) repair functions of ATM kinases promote telomere maintenance by inhibition of illegitimate recombination or fusion events between the legitimate ends of chromosomes and spontaneous DSBs. Drosophila will be an excellent model system for investigating how these ATM-dependent chromosome structural maintenance functions are deployed during development. Because neurons appear to be particularly sensitive to loss of ATM in both flies and humans, this system should be particularly useful for identifying cell-specific factors that influence sensitivity to loss of dATM and are relevant for understanding the human disease, ataxia-telangiectasia.     

Acetylcholinesterase function is dispensable for sensory neurite growth but is critical for neuromuscular synapse stability

The enzyme acetylcholinesterase (AChE) terminates synaptic transmission at cholinergic synapses by hydrolyzing the neurotransmitter acetylcholine. In addition, AChE is thought to play several 'non-classical' roles that do not require catalytic function. Most prominent among these is facilitation of neurite growth. Here, we report that the zebrafish zieharmonika (zim) locus encodes AChE. We show that one mutant zim allele is caused by a pre-mature stop codon, resulting in a truncated protein that lacks both the catalytic site and the carboxy-terminal neuritogenic domain. To explore the 'non-classical' role of AChE, we examined embryos mutant for this allele. In contrast to previous results using a catalytic-inactive allele, our analysis demonstrates that AChE is dispensable for muscle fiber development and Rohon-Beard sensory neuron growth and survival. Moreover, we show that in the absence of AChE, acetylcholine receptor clusters at neuromuscular junctions initially assemble, but that these clusters are not maintained. Taken together, our results demonstrate that AChE is dispensable for its proposed non-classical roles in muscle fiber formation and sensory neuron development, but is crucial for regulating the stability of neuromuscular synapses.     

ATR suppresses telomere fragility and recombination but is dispensable for elongation of short telomeres by telomerase

Telomere shortening caused by incomplete DNA replication is balanced by telomerase-mediated telomere extension, with evidence indicating that the shortest telomeres are preferred substrates in primary cells. Critically short telomeres are detected by the cellular DNA damage response (DDR) system. In budding yeast, the important DDR kinase Tel1 (homologue of ATM [ataxia telangiectasia mutated]) is vital for telomerase recruitment to short telomeres, but mammalian ATM is dispensable for this function. We asked whether closely related ATR (ATM and Rad3 related) kinase, which is important for preventing replicative stress and chromosomal breakage at common fragile sites, might instead fulfill this role. The newly created ATR-deficient Seckel mouse strain was used to examine the function of ATR in telomerase recruitment and telomere function. Telomeres were recently found to resemble fragile sites, and we show in this study that ATR has an important role in the suppression of telomere fragility and recombination. We also find that wild-type ATR levels are important to protect short telomeres from chromosomal fusions but do not appear essential for telomerase recruitment to short telomeres in primary mouse embryonic fibroblasts from the ATR-deficient Seckel mouse model. These results reveal a previously unnoticed role for mammalian ATR in telomere protection and stability.     

IgG1 is pathogenic in Leishmania mexicana infection

There are >2 million new cases of leishmaniasis annually, and no effective vaccine has been developed to prevent infection. In murine infection, Leishmania mexicana, which lives intracellularly in host macrophages, has developed pathways to hijack host IgG to induce a suppressive IL-10 response through FcγRs, the cell-surface receptors for IgG. To guide vaccine development away from detrimental Ab responses, which can accompany attempts to induce cell-mediated immunity, it is crucial to know which isotypes of IgG are pathogenic in this infection. We found that IgG1 and IgG2a/c induce IL-10 from macrophages in vitro equally well but through different FcγR subtypes: IgG1 through FcγRIII and IgG2a/c through FcγRI primarily, but also through FcγRIII. In sharp contrast, mice lacking IgG1 develop earlier and stronger IgG2a/c, IgG3, and IgM responses to L. mexicana infection and yet are more resistant to the infection. Thus, IgG1, but not IgG2a/c or IgG3, is pathogenic in vivo, in agreement with prior studies indicating that FcγRIII is required for chronic disease. This calls into question the assumption that macrophages, which should secrete IL-10 in response to IgG1 and IgG2a/c immune complexes, are the most important source of IL-10 generated by IgG-FcγR engagement in L. mexicana infection. Further investigations are required to better determine the cell type responsible for this immunosuppressive FcγRIII-induced IL-10 pathway and whether IgG2a/c is protective.     

O-GlcNAcase is essential for embryonic development and maintenance of genomic stability

Dysregulation of O-GlcNAc modification catalyzed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) contributes to the etiology of chronic diseases of aging, including cancer, cardiovascular disease, type 2 diabetes, and Alzheimer's disease. Here we found that natural aging in wild-type mice was marked by a decrease in OGA and OGT protein levels and an increase in O-GlcNAcylation in various tissues. Genetic disruption of OGA resulted in constitutively elevated O-GlcNAcylation in embryos and led to neonatal lethality with developmental delay. Importantly, we observed that serum-stimulated cell cycle entry induced increased O-GlcNAcylation and decreased its level after release from G2/M arrest, indicating that O-GlcNAc cycling by OGT and OGA is required for precise cell cycle control. Constitutively, elevated O-GlcNAcylation by OGA disruption impaired cell proliferation and resulted in mitotic defects with downregulation of mitotic regulators. OGA loss led to mitotic defects including cytokinesis failure and binucleation, increased lagging chromosomes, and micronuclei formation. These findings suggest an important role for O-GlcNAc cycling by OGA in embryonic development and the regulation of the maintenance of genomic stability linked to the aging process.     

Fission yeast Rhp51 is required for the maintenance of telomere structure in the absence of the Ku heterodimer

The Schizosaccharomyces pombe Ku70–Ku80 heterodimer is required for telomere length regulation. Lack of pku70⁺ results in telomere shortening and striking rearrangements of telomere-associated sequences. We found that the rearrangements of telomere-associated sequences in pku80⁺ mutants are Rhp51 dependent, but not Rad50 dependent. Rhp51 bound to telomere ends when the Ku heterodimer was not present at telomere ends. We also found that the single-stranded G-rich tails increased in S phase in wild-type strains, while deletion of pku70⁺ increased the single-stranded overhang in both G₂ and S phase. Based on these observations, we propose that Rhp51 binds to the G-rich overhang and promotes homologous pairing between two different telomere ends in the absence of Ku heterodimer. Moreover, pku80 rhp51 double mutants showed a significantly reduced telomere hybridization signal. Our results suggest that, although Ku heterodimer sequesters Rhp51 from telomere ends to inhibit homologous recombination activity, Rhp51 plays important roles for the maintenance of telomere ends in the absence of the Ku heterodimer.     

Hoogsteen G-G base pairing is dispensable for telomere healing in yeast.

The G-rich strands of most eukaryotic telomeres are capable of forming highly folded structures in vitro, mediated, in part, through Hoogsteen G-G base pairing. The ability of most telomeres to form these structures has led to the suggestion that they play an important role in telomere addition. I have investigated this possibility in the yeast Saccharomyces cerevisiae through the use of an in vivo assay that measures healing via poly(G1-3T) addition onto plasmid substrates containing synthetic telomeres. Synthetic telomere healing is a highly size- and sequence-specific process that allows the discrimination of telomeres of differing efficiency. Plasmids containing synthetic telomeres with differing abilities to form secondary structures were tested in this assay for healing in vivo. The results of this study demonstrate that telomeres incapable of forming Hoogsteen base pairs nonetheless serve as efficient substrates for poly(G1-3T) addition, indicating that intramolecular Hoogsteen G-G base pairing is not essential for this process.     

Ku80 gene is related to non-homologous end-joining and genome stability in Aspergillus niger

In this study, the ku70 and ku80 homologs from the Aspergillus niger genome were identified and their function was analyzed using targeted mutagenesis. The role of the ku80 gene in non-homologous end-joining (NHEJ) was investigated by calculating the frequency of homologous recombination. The transformation test verified that the frequency of homologous recombination significantly increased, from 1.78 to 65.6% in ku80 single deletion strains and to 100% in ku70/ku80 double deletion strains. These results suggest that the ku80 gene is important for non-homologous end-joining. Although the morphology of the ku deletion strains colonies was similar to that of the wildtype strain, mutants were more sensitive to the mutagen phleomycin. Furthermore, the purified ku80 deletion strain produced some sectored colonies on hygromycin B-containing plates. This result suggests that the ku80 gene deletion leads to genomic instability in A. niger.     

Ku80 plays a role in non-homologous recombination but is not required for T-DNA integration in Arabidopsis

Chromosomal breaks are repaired by homologous recombination (HR) or non-homologous end joining (NHEJ) mechanisms. The Ku70/Ku80 heterodimer binds DNA ends and plays roles in NHEJ and telomere maintenance in organisms ranging from yeast to humans. We have previously identified a ku80 mutant of the model plant Arabidopsis thaliana and shown the role of Ku80 in telomere homeostasis in plant cells. We show here that this mutant is hypersensitive to the DNA-damaging agent methyl methane sulphonate and has a reduced capacity to carry out NHEJ recombination. To understand the interplay between HR and NHEJ in plants, we measured HR in the absence of Ku80. We find that the frequency of intrachromosomal HR is not affected by the absence of Ku80. Previous work has clearly implicated the Ku heterodimer in Agrobacterium-mediated T-DNA transformation of yeast. Surprisingly, ku80 mutant plants show no defect in the efficiency of T-DNA transformation of plants with Agrobacterium, showing that an alternative pathway must exist in plants.     

Arsenic-induced sumoylation of Mus81 is involved in regulating genomic stability

Chronic environmental exposure to metal toxicants such as chromium and arsenic is closely related to the development of several types of common cancers. Genetic and epigenetic studies in the past decade reveal that post-translational modifications of histones play a role in metal carcinogenesis. However, exact molecular mechanisms of metal carcinogenesis remain to be elucidated. In this study we found that As₂O₃, an environmental metal toxicant, upregulated overall modifications of many cellular proteins by SUMO2/3. Sumoylated proteins from arsenic-treated cells constitutively expressing His₆-SUMO2 were pulled down by Ni-IDA resin under denaturing conditions. Mass spectrometric analysis revealed over 100 proteins that were potentially modified by sumoylation. Mus81, a DNA endonuclease involved in homologous recombination repair, was among the identified proteins whose sumoylation was increased after treatment with As₂O_3. We further showed that K10 and K524 were 2 lysine residues essential for Mus81 sumoylation. Moreover, we demonstrated that Mus81 sumoylation is important for normal mitotic chromosome congression and that cells expressing SUMO-resistant Mus81 mutants displayed compromised DNA damage responses after exposure to metal toxins such as Cr(VI) and arsenic.     

Disruption of mannose activation in Leishmania mexicana: GDP-mannose pyrophosphorylase is required for virulence, but not for viability.

In eukaryotes, the enzyme GDP-mannose pyrophosphorylase (GDPMP) is essential for the formation of GDP-mannose, the central activated mannose donor in glycosylation reactions. Deletion of its gene is lethal in fungi, most likely as a consequence of disrupted glycoconjugate biosynthesis. Furthermore, absence of GDPMP enzyme activity and the expected loss of all mannose-containing glycoconjugates have so far not been observed in any eukaryotic organism. In this study we have cloned and characterized the gene encoding GDPMP from the eukaryotic protozoan parasite Leishmania mexicana. We report the generation of GDPMP gene deletion mutants of this human pathogen that are devoid of detectable GDPMP activity and completely lack mannose-containing glycoproteins and glycolipids, such as lipophosphoglycan, proteophosphoglycans, glycosylphosphatidylinositol protein membrane anchors, glycoinositolphospholipids and N-glycans. The loss of GDPMP renders the parasites unable to infect macrophages or mice, while gene addback restores virulence. Our study demonstrates that GDP-mannose biosynthesis is not essential for Leishmania viability in culture, but constitutes a virulence pathway in these human pathogens.