Long interspersed element-1 (LINE-1 or L1) elements are abundant, non-long-terminal-repeat (non-LTR) retrotransposons that comprise ∼17% of human DNA1. The average human genome contains ∼80–100 retrotransposition-competent L1s (ref. 2), and they mobilize by a process that uses both the L1 endonuclease and reverse transcriptase, termed target-site primed reverse transcription3,4,5. We have previously reported an efficient, endonuclease-independent L1 retrotransposition pathway (ENi) in certain Chinese hamster ovary (CHO) cell lines that are defective in the non-homologous end-joining (NHEJ) pathway of DNA double-strand-break repair6. Here we have characterized ENi retrotransposition events generated in V3 CHO cells, which are deficient in DNA-dependent protein kinase catalytic subunit (DNA-PKcs) activity and have both dysfunctional telomeres and an NHEJ defect. Notably, ∼30% of ENi retrotransposition events insert in an orientation-specific manner adjacent to a perfect telomere repeat (5′-TTAGGG-3′). Similar insertions were not detected among ENi retrotransposition events generated in controls or in XR-1 CHO cells deficient for XRCC4, an NHEJ factor that is required for DNA ligation but has no known function in telomere maintenance. Furthermore, transient expression of a dominant-negative allele of human TRF2 (also called TERF2) in XRCC4-deficient XR-1 cells, which disrupts telomere capping, enables telomere-associated ENi retrotransposition events. These data indicate that L1s containing a disabled endonuclease can use dysfunctional telomeres as an integration substrate. The findings highlight similarities between the mechanism of ENi retrotransposition and the action of telomerase, because both processes can use a 3′ OH for priming reverse transcription at either internal DNA lesions or chromosome ends7,8. Thus, we propose that ENi retrotransposition is an ancestral mechanism of RNA-mediated DNA repair associated with non-LTR retrotransposons that may have been used before the acquisition of an endonuclease domain.
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We thank A. M. DesLauriers for help with flow cytometry and cell sorting; R. Lyons for help with sequencing; T. de Lange for discussions and the dominant-negative TRF2 expression construct; M. Abe for the murine DNA-PKcs expression construct; G. Hammer and T. Else for help with the metaphase analysis; and C. Greider for discussions and use of equipment and reagents for immunocytochemistry experiments. We thank T. Else, T. Glover, T. Glaser, N. Howlett, T. Wilson and current members of the Moran laboratory for discussions during the course of this work. T.A.M. was supported in part by a NIH training grant and is now a Leukemia and Lymphoma Society Fellow. J.L.G.-P. was supported in part by a MEC/Fulbright postdoctoral grant. G.E.T. is partially supported by a grant from the Human Frontier Science Program. T.D.S was supported in part by a grant from the National Institutes of Health. J.S. was supported in part by a grant from the Pew Foundation. J.V.M was supported in part by grants from the W. M. Keck Foundation and the National Institutes of Health.
Author Contributions T.A.M is co-first author and is a corresponding author. She contributed to the original concept, designed and performed experiments, analysed the data, and wrote the first draft of the manuscript. J.L.G.-P. is co-first author. He contributed to the concept, designed and performed experiments, analysed the data, and helped write and revise the manuscript. T.D.S and G.E.T. contributed reagents, helped with data interpretation and provided helpful comments during the course of this study. J.S. contributed the murine DNA-PKcs complemented cell lines, performed functional analyses on those cell lines, helped with data analysis, and helped revise the manuscript. J.V.M. is the senior author. He contributed to the original concept, analysed the data, revised the manuscript and provided financial support.
Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.
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Morrish, T., Garcia-Perez, J., Stamato, T. et al. Endonuclease-independent LINE-1 retrotransposition at mammalian telomeres. Nature 446, 208–212 (2007). https://doi.org/10.1038/nature05560
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