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

Trans-Bilayer Ion Conduction by Proline Containing Cyclic Hexapeptides and Effects of Amino Acid Substitutions on Ion Conducting Properties

Several ion channel forming cyclic peptides have been reported over the past two decades and various ion conducting mechanisms have been proposed. In this article, we report on amino acid substitutions in cyclic hexapeptides and their effects on the ion conducting properties of these peptides. Cyclic hexapeptides, cyclo(Pro-Xxx-Yyy)2, containing two Pro residues, were used as the main framework. The substitution is performed at the Xxx positions with cationic/hydrophilic Lys or hydrophobic Leu. Yyy positions were substituted with D-Phe, D-Ala, or Gly. The peptides which were absent Lys residues showed ion conducting profiles with clear transitions of electric currents, whereas the peptides containing Lys residues tended to exhibit spiky or burst-like profiles. These profiles were altered single state profiles by the protection of ε-amino groups with aromatic protecting groups. The protected analogs exhibited significant decrease in ion conductance. These results indicated that peptides containing Lys conduct ions without forming ring stacked tube-like structure. Ion channel properties were also affected by conformational changes of the cyclic peptides induced by substitution of the Yyy positions. Enhancement of intramolecular β-turn structures of cyclic peptides tended to decrease their ion conductance values

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

Characterization of the sequence specificity of

the R1Bm endonuclease domain by structural and biochemical studie

Small-angle X-ray scattering study on CEL-III, a hemolytic lectin from Holothuroidea Cucumaria echinata, and its oligomer induced by the binding of specific carbohydrate

AbstractHemolytic lectin CEL-III from a marine invertebrate Cucumaria echinata forms an oligomer upon binding of specific carbohydrate such as lactose at high pH values and in the presence of high concentrations of salt. In this study, using small-angle X-ray scattering, we characterized CEL-III and its oligomer induced by the binding of lactose. The molecular mass of the oligomer was determined as 1019 kDa from its forward scattering value, compared with 47 490 Da for the monomer. This oligomer size is much larger than that estimated using SDS–polyacrylamide gel electrophoresis (SDS-PAGE, 270 kDa). The monomer has a 24.6 Å radius of gyration and can be approximated by a rod which has a 20 Å radius and a height of 75 Å, while the oligomer has a 101.4Å radius of gyration. Together with the comparison of the radii of gyration and the forward scattering of the cross-section of the monomer and oligomer, it is suggested that in aqueous solution the oligomer comprises three or four molecules of a smaller unit which was observed by SDS-PAGE (270 kDa), held by a relatively weak interaction. The scattering profile also suggests that the oligomer has a hole in its central axis which might be associated with the formation of ion-permeable pores in the erythrocyte membrane by CEL-III during the hemolytic process