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Molecular and Cellular Biology, January 2001, p. 100-108, Vol. 21, No. 1
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.1.100-108.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Sequence-Specific Recognition and Cleavage of
Telomeric Repeat (TTAGG)n by Endonuclease of
Non-Long Terminal Repeat Retrotransposon TRAS1
Tomohiro
Anzai,
Hidekazu
Takahashi, and
Haruhiko
Fujiwara*
Department of Integrated Biosciences,
Graduate School of Frontier Sciences, University of Tokyo,
Bunkyo-ku, Tokyo 113-0033, Japan
Received 13 March 2000/Returned for modification 4 May
2000/Accepted 13 October 2000
The telomere of the silkworm Bombyx mori consists of
(TTAGG/CCTAA)n repeats and harbors a large
number of telomeric repeat-specific non-long terminal repeat
retrotransposons, such as TRAS1 and SART1. To understand how these
retrotransposons recognize and integrate into the telomeric repeat in a
sequence-specific manner, we expressed the apurinic-apryrimidinic
endonuclease-like endonuclease domain of TRAS1 (TRAS1 EN), which is
supposed to digest the target DNA, and characterized its enzymatic
properties. Purified TRAS1 EN could generate specific nicks on both
strands of the telomeric repeat sequence between T and A of the
(TTAGG)n strand (bottom strand) and between C
and T of the (CCTAA)n strand (top strand).
These sites are consistent with insertion sites expected from the
genomic structure of boundary regions of TRAS1. Time course studies of
nicking activities on both strands revealed that the cleavages on the
bottom strand preceded those on the top strand, supporting the
target-primed reverse transcription model. TRAS1 EN could cleave the
telomeric repeats specifically even if it was flanked by longer tracts
of nontelomeric sequence, indicating that the target site specificity
of the TRAS1 element was mainly determined by its EN domain. Based on
mutation analyses, TRAS1 EN recognizes less than 10 bp around the
initial cleavage site (upstream 7 bp and downstream 3 bp), and the
GTTAG sequence especially is essential for the cleavage reaction on the
bottom strand (5'. . . TTAGGTT
AGG . . . 3'). TRAS1 EN, the first identified endonuclease digesting telomeric
repeats, may be used as a genetic tool to shorten the telomere in
insects and some other organisms.
*
Corresponding author. Mailing address: Department of
Integrated Biosciences, Graduate School of Frontier Sciences,
University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan.
Phone and Fax: 81-3-5841-4447. E-mail:
haruh{at}k.u-tokyo.ac.jp.
Molecular and Cellular Biology, January 2001, p. 100-108, Vol. 21, No. 1
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.1.100-108.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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