Sequence-Specific Recognition and Cleavage of Telomeric Repeat (TTAGG)_nby Endonuclease of Non-Long Terminal Repeat Retrotransposon TRAS1

Abstract

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 2 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.

Crystal Structure of the Endonuclease Domain Encoded by the Telomere-specific Long Interspersed Nuclear Element, TRAS1

“…Mutagenesis of TRAS1-EN-The H237A mutant was constructed in a previous study (16). The point mutations were generated by a QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions.…”

“…Protein Purification and Crystallization-The TRAS1-EN expression vector (pHisT1EN) was constructed and transfected into E. coli BL21(DE3)/pLysS strain as described previously (16). The His 10 -tagged TRAS1-ENs were purified by Ni 2ϩ -bound HiTrap chelating column (Amersham Biosciences), followed by a Factor Xa digestion.…”

“…Oligonucleotide Cleavage-32 P-labeled telomeric DNA was prepared as described previously (16). The 40-bp oligonucleotide containing 28 S rDNA sequence of the silkworm (5Ј-ACGAGATTCCCACTGTCCCTAT-CTACTATCTAGCGAAACC-3Ј) was also radiolabeled, annealed with the nonlabeled complementary strand, and gel-purified.…”

“…1A) (16). To confirm the sequence specificity of TRAS1-EN, its endonucleolytic activity was tested for various telomeric repeats from several organisms using double-stranded oligonucleotide substrates.…”

“…In addition, it is hypothesized that LINE-ENs primarily cleave the bottom strand (e.g. TTAGG for TRAS1) followed by the top strand (CCTAA), supporting the target-primed reverse transcription model, which is a system unique to LINEs (13,[15][16][17]. At present, however, little is known about the mechanisms of sequence recognition and digestion by endonuclease domains during the process of targetprimed reverse transcription.…”