Abstract
BackgroundThe 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 in vivo has not so far been demonstrated. In addition, there is lack of information on the cellular effects of telomere shortening in human cells.ResultsHere, 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 in vitro assay demonstrated that the TRAS1EN-TRF1 chimeric endonucleases (T-EN and EN-T) cut the human (TTAGGG)n repeats specifically. The concentration of TRAS1EN-TRF1 chimeric endonucleases necessary for the cleavage of (TTAGGG)n repeats 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.ConclusionsA novel TRAS1EN-TRF1 chimeric endonuclease (EN-T) cuts the human telomeric repeats (TTAGGG)n specifically in vitro and in vivo. Thus, the chimeric endonuclease which is expressed from an adenoviral vector can suppress cell proliferation of cancer cells.
Highlights
The ends of chromosomes, termed telomeres consist of repetitive DNA
Construction and expression of chimeric endonucleases In order to increase the cleavage activity for human telomeric TTAGGG sequences, we constructed several types of chimeric endonuclease versions of TRAS1EN (EN), which cuts the silkworm TTAGG repeats by fusing wildtype TRAS1EN with various parts of TRF1 (T) either at the N- or C-terminal ends of TRAS1EN (Figure 1a)
We made chimeric endonuclease-like domain (EN) constructs with a full-length sequence of TRF1 (TEN, EN-T): only the TRF1-Myb component (M-EN, ENM) and with the nuclear localization signal (NLS) to Myb region of TRF1 (EN-NM)
Summary
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. The DNA component of telomeres, typically 5-8 kb long, consists of tandem arrays of short, repetitive G-rich sequences--TTAGGG in vertebrates--oriented 5'- to -3' towards the end of the chromosome [2]. In order to induce this rapidly, we investigated a novel method of shortening telomeres with the endonuclease domain of a telomere-specific non-long terminal repeat (LTR) retrotransposon, TRAS1. Non-LTR retrotransposons, known as long interspersed nuclear elements (LINEs), are transposable elements that insert into the genome via RNA intermediates. Most such transposable elements integrate into the genome randomly, some LINEs have very restricted target sequences for integration [7]. If TRAS1EN could cut telomeres in human cancer cells and effectively, we hypothesized that the proliferation of cancer cells would be inhibited
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