2002 Nucleic Acids Research, 2005, Vol. 33, No. 6 2H209A mutant, which contains a non-sense mutation in the catalytic motif of EN (Figure 3A). This mutation has been shown to severely decrease its endonucleolytic activity of R1-EN in vitro (Aoyagi and Fujiwara, unpublished data), and the primary sequence around the hotspot ( 180) showed no similarity with that of the speciﬁc cleavage site by R1-EN in vitro (15), indicating that this integration was not resulted from the intrinsic EN activity of R1Bm. The position 180 was only site for integration other than the R1-intrinsic target (four clones in Figure 3B and C) and their reverse transcription started various regions of template RNA, suggesting that the 180 position was precedently formed in 28S rDNA before the EN of R1Bm works during TPRT reaction. A low level of retrotransposition by human L1 which lacks its endonuclease activity was reported in vivo and in vitro (31,32). This suggests that such L1 integrations occurred at the pre-formed double- strand break on the chromosomal DNA. Although the mech- anism to generate a nick on this hot spot ( 180) was unclear, the DNA cleavage might be pre-formed possibly by the recombination event around this spot. ACKNOWLEDGEMENTS We thank K.K. Kojima for critical reading of the manuscript. This work was supported by grants from the Ministry of Education, Science and Culture of Japan (MESCJ) and by a Grant-in-Aid from the Research for the Future Program of the Japan Society for the Promotion Science (JSPS). T.A. and M.O. are recipients of Research fellowships of the JSPS for Young Scientists. Funding to pay the Open Access publication charges for this article was provided by MESCJ. Conflict of interest statement. None declared. REFERENCES 1. Arkhipova,I. and Meselsonm,M. (2000) Transposable elements in sexual and ancient asexual taxa. Proc. Natl Acad. Sci. USA, 97, 14473–14477. 2. Lander,E.S., Linton,L.M., Birren,B., Nusbaum,C., Zody,M.C., Baldwin,J., Devon,K., Dewar,K., Doyle,M., FitzHugh,W. et al. (2001) Initial sequencing and analysis of the human genome. Nature, 409, 860–921. 3. Moran,J.V., DeBerardinis,R.J. and Kazazian,H.H.,Jr (1999) Exon shuffling by L1 retrotransposition. Science, 283, 1530–1534. 4. Gilbert,N., Lutz-Prigge,S. and Moran,J.V. (2002) Genomic deletions created upon LINE-1 retrotransposition. Cell, 110, 315–325. 5. Symer,D.E., Connelly,C., Szak,S.T., Caputo,E.M., Cost,G.J., Parmigiani,G. and Boeke,J.D. (2002) Human L1 retrotransposition is associated with genetic instability in vivo. Cell, 110, 327–338. 6. Malik,H.S., Burke,W.D. and Eickbush,T.H. (1999) The age and evolution of non-LTR retrotransposable elements. Mol. Biol. Evol., 16, 793–805. 7. Burke,W.D., Calalang,C.C. and Eickbush,T.H. (1987) The site-specific ribosomal insertion element type II of Bombyx mori (R2Bm) contains the coding sequence for a reverse transcriptase-like enzyme. Mol. Cell Biol., 7, 2221–2230. 8. Yang,J., Malik,H.S. and Eickbush,T.H. (1999) Identification of the endonuclease domain encoded by R2 and other site-specific, non-long terminal repeat retrotransposable elements. Proc. Natl Acad. Sci. USA, 96, 7847–7852. 9. Xiong,Y. and Eickbush,T.H. (1988) The site-specific ribosomal DNA insertion element R1Bm belongs to a class of non-long-terminal-repeat retrotransposons. Mol. Cell Biol., 8, 114–123. 10. Luan,D.D. and Eickbush,T.H. (1995) RNA template requirements for target DNA-primed reverse transcription by the R2 retrotransposable element. Mol. Cell Biol., 15, 3882–3891. 11. Luan,D.D., Korman,M.H., Jakubczak,J.L. and Eickbush,T.H. (1993) Reverse transcription of R2Bm RNA is primed by a nick at the chromosomal target site: a mechanism for non-LTR retrotransposition. Cell, 72, 595–605. 12. Long,E.O. and Dawid,I.B. (1979) Expression of ribosomal DNA insertions in Drosophila melanogaster. Cell, 18, 1185–1196. 13. Kidd,S.J. and Glover,D.M. (1981) Drosophila melanogaster ribosomal DNA containing type II insertions is variably transcribed in different strains and tissues. J. Mol. Biol., 151, 645–662. 14. Luan,D.D. and Eickbush,T.H. (1996) Downstream 28S gene sequences on the RNA template affect the choice of primer and the accuracy of initiation by the R2 reverse transcriptase. Mol. Cell Biol., 16, 4726–4734. 15. Feng,Q., Schumann,G. and Boeke,J.D. (1998) Retrotransposon R1Bm endonuclease cleaves the target sequence. Proc. Natl Acad. Sci. USA, 95, 2083–2088. 16. Moran,J.V., Holmes,S.E., Naas,T.P., DeBerardinis,R.J., Boeke,J.D. and Kazazian,H.H.,Jr (1996) High frequency retrotransposition in cultured mammalian cells. Cell, 87, 917–927. 17. Takahashi,H. and Fujiwara,H. (2002) Transplantation of target site specificity by swapping the endonuclease domains of two LINEs. EMBO J., 21, 408–417. 18. Pickeral,O.K., Makalowski,W., Boguski,M.S. and Boeke,J.D. (2000) Frequent human genomic DNA transduction driven by LINE-1 retrotransposition. Genome Res., 10, 411–415. 19. Goodier,J.L., Ostertag,E.M. and Kazazian,H.H.,Jr (2000) Transduction of 30-flanking sequences is common in L1 retrotransposition. Hum. Mol. Genet., 9, 653–657. 20. Osanai,M., Takahashi,H., Kojima,K.K., Hamada,M. and Fujiwara,H. (2004) Essential motifs in the 30 untranslated region required for retrotransposition and the precise start of reverse transcription in non-long-terminal-repeat retrotransposan SART1. Mol. Cell Biol., 24, 7902–7913. 21. Jakubczak,J.L., Burke,W.D. and Eickbush,T.H. (1991) Retrotransposable elements R1 and R2 interrupt the rRNA genes of most insects. Proc. Natl Acad. Sci. USA, 88, 3295–3299. 22. Fujiwara,H., Ogura,T., Takada,N., Miyajima,N., Ishikawa,H. and Maekawa,H. (1984) Introns and their flanking sequences of Bombyx mori rDNA. Nucleic Acids Res., 12, 6861–6869. 23. Ergun,S., Buschmann,C., Heukeshoven,J., Dammann,K., Schnieders,F., Lauke,H., Chalajour,F., Kilic,N., Stratling,W.H. and Schumann,G.G. (2004) Cell type-specific expression of LINE-1 open reading frames 1 and 2 in fetal and adult human tissues. J. Biol. Chem., 279, 27753–27763. 24. Ostertag,E.M. and Kazazian,H.H.,Jr (2001) Biology of mammalian L1 retrotransposons. Annu. Rev. Genet., 35, 501–538. 25. Perez-Gonzalez,C.E. and Eickbush,T.H. (2002) Rates of R1 and R2 retrotransposition and elimination from the rDNA locus of Drosophila melanogaster. Genetics, 162, 799–811. 26. Fujimoto,H., Hirukawa,Y., Tani,H., Matsuura,Y., Hashido,K., Tsuchida,K., Takada,N., Kobayashi,M. and Maekawa,H. (2004) Integration of the 50 end of the retrotransposon, R2Bm, can be complemented by homologous recombination. Nucleic Acids Res., 32, 1555–1565. 27. Kojima,K.K. and Fujiwara,H. (2003) Evolution of target specificity in R1 clade non-LTR retrotransposons. Mol. Biol. Evol., 20, 351–361. 28. Anzai,T., Takahashi,H. and Fujiwara,H. (2001) Sequence-specific recognition and cleavage of telomeric repeat (TTAGG)(n) by endonuclease of non-long terminal repeat retrotransposon TRAS1. Mol. Cell Biol., 21, 100–108. 29. Eickbush,D.G., Luan,D.D. and Eickbush,T.H. (2000) Integration of Bombyx mori R2 sequences into the 28S ribosomal RNA genes of Drosophila melanogaster. Mol. Cell Biol., 20, 213–223. 30. Feng,Q., Moran,J.V., Kazazian,H.H.,Jr and Boeke,J.D. (1996) Human L1 retrotransposon encodes a conserved endonuclease required for retrotransposition. Cell, 87, 905–916. 31. Morrish,T.A., Gilbert,N., Myers,J.S., Vincent,B.J., Stamato,T.D., Taccioli,G.E., Batzer,M.A. and Moran,J.V. (2002) DNA repair mediated by endonuclease-independent LINE-1 retrotransposition. Nature Genet., 31, 159–165. 32. Cost,G.J., Feng,Q., Jacquier,A. and Boeke,J.D. (2002) Human L1 element target-primed reverse transcription in vitro. EMBO J., 21, 5899–5910.