The telomere-specific long interspersed nuclear element, TRAS1, encodes an endonuclease domain, TRAS1-EN, which specifically cleaves the telomeric repeat targets (TTAGG) n of insects and (TTAGGG) n of vertebrates. To elucidate the sequence-specific recognition properties of TRAS1-EN, we determined the crystal structure at 2.4-Å resolution. TRAS1-EN has a four-layered ␣/␤ sandwich structure; its topology is similar to apurinic/ apyrimidinic endonucleases, but the ␤-hairpin (␤ 10 -␤ 11 ) at the edge of the DNA-binding surface makes an extra loop that distinguishes TRAS1-EN from cellular apurinic/apyrimidinic endonucleases. A protein-DNA complex model suggests that the ␤ 10 -␤ 11 hairpin fits into the minor groove, enabling interaction with the telomeric repeats. Mutational studies of TRAS1-EN also indicated that the Asp-130 and ␤ 10 -␤ 11 hairpin structure are involved in specific recognition of telomeric repeats.Non-long terminal repeat retrotransposons, also known as long interspersed nuclear elements (LINEs), 1 are transposable elements that encode a reverse transcriptase and insert into genomic locations via RNA intermediates. The recent progress of the human genome project has revealed that one LINE, L1, integrates throughout chromosomes and occupies more than 20% of the genome (1). The integration of L1 may play a role in genetic diseases and cancers (2) and in gene evolution and genome reconstruction (3-5).The LINEs have been classified into two types according to the number of open reading frames (ORFs) (6). The first type of element has a single ORF and encodes an endonuclease domain near its C terminus; this type of endonuclease (also known as restriction enzyme-like endonuclease) is similar in some residual motifs to various prokaryotic restriction enzymes (7). The second type of element has two ORFs; ORF1 encodes a retroviral Gag-like protein whose function is still unclear, and ORF2 encodes a protein with an endonuclease domain at its N terminus and a reverse transcriptase domain in the center of the ORF. This class of endonuclease domain is made up of about 250 amino acid residues and shares sequence homology with apurinic/apyrimidinic endonucleases (APE), such as human APE1 and Escherichia coli exonuclease III.Most of the APE-like endonuclease-encoding retrotransposons do not insert in a sequence-specific manner into the host genome, like the human L1 elements that cleave AT-rich sequences with a low sequence specificity (8). However, several endonuclease-encoding LINE have very restricted integration targets within the genome (9). TRAS1 and R1Bm, found in Bombyx mori, are the typical sequence-specific elements, which insert between T and A of the (TTAGG) n telomeric repeat and a specific site in 28 S rDNA of B. mori, respectively (10, 11). Recent studies have shown that the endonuclease domain in the ORF2 of TRAS1 (12) and R1Bm (13) determines its own target sequence recognition and DNA cleavage activity.The amino acid identity between human APE1 and the endonuclease domain of TRAS1 (TRAS1-EN) is n...

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.