Creation of a novel telomere-cutting endonuclease based on the EN domain of telomere-specific non-long terminal repeat retrotransposon, TRAS1

<p>Abstract</p> <p>Background</p> <p>The ends of chromosomes, termed telomeres consist of repetitive DNA. The telomeric sequences shorten with cell division and, when telomeres are critically abbreviated, cells stop proliferating. However, in cancer cells, by the expression of telomerase which elongates telomeres, the cells can continue proliferating. Many approaches for telomere shortening have been pursued in the past, but to our knowledge, cutting telomeres <it>in vivo </it>has not so far been demonstrated. In addition, there is lack of information on the cellular effects of telomere shortening in human cells.</p> <p>Results</p> <p>Here, we created novel chimeric endonucleases to cut telomeres by fusing the endonuclease domain (TRAS1EN) of the silkworm's telomere specific non-long terminal repeat retrotransposon TRAS1 to the human telomere-binding protein, TRF1. An <it>in vitro </it>assay demonstrated that the TRAS1EN-TRF1 chimeric endonucleases (T-EN and EN-T) cut the human (TTAGGG)_nrepeats specifically. The concentration of TRAS1EN-TRF1 chimeric endonucleases necessary for the cleavage of (TTAGGG)_nrepeats was about 40-fold lower than that of TRAS1EN alone. When TRAS1EN-TRF1 endonucleases were introduced into human U2OS cancer cells using adenovirus vectors, the enzymes localized at telomeres of nuclei, cleaved and shortened the telomeric DNA by double-strand breaks. When human U2OS and HFL-1 fibroblast cells were infected with EN-T recombinant adenovirus, their cellular proliferation was suppressed for about 2 weeks after infection. In contrast, the TRAS1EN mutant (H258A) chimeric endonuclease fused with TRF1 (ENmut-T) did not show the suppression effect. The EN-T recombinant adenovirus induced telomere shortening in U2OS cells, activated the p53-dependent pathway and caused the senescence associated cellular responses, while the ENmut-T construct did not show such effects.</p> <p>Conclusions</p> <p>A novel TRAS1EN-TRF1 chimeric endonuclease (EN-T) cuts the human telomeric repeats (TTAGGG)_nspecifically <it>in vitro </it>and <it>in vivo</it>. Thus, the chimeric endonuclease which is expressed from an adenoviral vector can suppress cell proliferation of cancer cells.</p

Characterization of the sequence specificity of the R1Bm endonuclease domain by structural and biochemical studies

R1Bm is a long interspersed element (LINE) inserted into a specific sequence within 28S rDNA of the silkworm genome. Of two open reading frames (ORFs) of R1Bm, ORF2 encodes a reverse transcriptase (RT) and an endonuclease (EN) domain which digests specifically both top and bottom strand of the target sequence in 28S rDNA. To elucidate the sequence specificity of EN domain of R1Bm (R1Bm EN), we examined the cleavage tendency for the target sequences, and found that 5′-A(G/C)(A/T)!(A/G)T-3′ is the consensus sequence (! = cleavage site). We also determined the crystal structure of R1Bm EN at 2.0 Å resolution. Its structure was basically similar to AP endonuclease family, but had a special β-hairpin at the edge of the DNA binding surface, which is a common feature among EN of LINEs. Point-mutations on the DNA binding surface of R1Bm EN significantly decreased the cleavage activities, but did not affect the sequence recognition in most residues. However, two mutants Y98A and N180A had altered cleavage patterns, suggesting an important role of these residues (Y98 and N180) for the sequence recognition of R1Bm EN. In addition, Y98A mutant showed another cleavage pattern, that implies de novo design of novel sequence-specific EN

Unprecedented glucose production using auto-catalytic hydrothermal hydrolysis of lignocellulosic biomass with no catalyst addition

We propose a novel and simple glucose production method involving autocatalytic hydrothermal hydrolysis of biomass without catalyst additives. This method consists of air-oxidation at a relatively lower temperature than that of the conventional method and subsequent hydrothermal hydrolysis. Japanese cedar (indigestible softwood) was initially air–oxidized by heating at 180–200 °C for 3 h. This induced the decomposition of a small portion of cellulose in the cedar sample, although 93.4% of cellulose remained at 200 °C. The functional acidic groups (phenol, lactonic, and carboxy groups) specifically increased during the initial air–oxidation. Following this, the acidic groups autocatalytically accelerated hydrothermal cellulose hydrolysis, resulting in a maximum glucose yield of 139.6 mg/g raw material at a severity factor (R0) of 3.2 × 104. Keywords: Cellulose hydrolysis, Oxidation, Glucose production, Hot compressed water, Hydrothermal treatmen

Characterization of the sequence specificity of

the R1Bm endonuclease domain by structural and biochemical studie

Structures and Properties of Saturn-Like Complexes Composed of Oligothiophene Macrocycle with Methano[60]fullerene and [70]Fullerene

Ď -Expanded oligothienylene macrocycle with a large inner cavity incorporates fullerenes such as methano[60]fullerene (C61H2) and [70]fullerene (C70) inside to form Saturn-like complexes. Although the oligothiophene macrocycle weakly interacts with fullerenes in solution, it forms stable Saturn-like fullerene complexes in the solid state. X-ray analysis of the Saturn-like complexes exhibited short contacts between the sulfur atoms of the oligothiophene macrocycle and fullerene-carbons, which hinder the rotation of fullerenes. As a result, the non-covalent interaction between the oligothiophene macrocycle and fullerenes was employed in crystal structure determination of fullerenes. UV-vis-NIR spectra of the Saturn-like complexes showed weak donor-acceptor interaction between the oligothiophene macrocycle and fullerenes.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author