Journal of Molecular Biology
The Human LINE-1 Retrotransposon Creates DNA Double-strand Breaks
Introduction
Long interspersed element-1 (L1) is an active, autonomous family of retroelements that comprises 17% of the human genome.1 Another 11% of the human genome is composed of Alu and SVA elements that require L1 for mobilization in trans.2, 3, 4 L1 utilizes an APE-like endonuclease to insert into the target DNA.5 On the basis of in vitro analysis of the R2 element from the silkworm Bombyx mori (R2Bm), the current model for non-long terminal repeat (non-LTR) element insertion is target-primed reverse transcription (TPRT) (Figure 1).6, 7 A variation of this model that incorporates the frequent creation of target-site duplications (TSDs) also likely explains the majority of human genomic and de novo L1 insertions.8, 9, 10, 11 In these models of TPRT, cleavage of both strands of genomic DNA is required, which implies that an intermediate equivalent to a DSB is present during the integration process.
Recent studies have begun to recognize the interplay between mobile elements and DNA repair.12, 13, 14 As with retroviruses and transposons, which have well-characterized insertion mechanisms, host DNA repair enzymes are likely to both contribute to, and suppress, L1 retrotransposition by recognizing and processing the retrotransposon-initiated nicks or breaks. In contrast to transposons and retroviruses, with well-established in vitro models of the insertion process, molecular dissection of TPRT intermediates has been demonstrated only partially.6, 7, 15 Several other non-LTR retrotransposon systems have demonstrated a relationship between these elements, genetic instability, and host repair enzymes. Group II introns in yeast mitochondria induce DSBs in DNA16 and display hallmarks of DSB repair processing after successful integration,17, 18 although the genetic requirements have not been demonstrated in that system. The L1.LtrB intron from the Lactococcus lactis bacterium has been shown in molecular assays to utilize TPRT as a subpathway for insertion,19, 20 and it has been demonstrated recently to require host (Escherichia coli) repair proteins for later steps in integration.21 Other than L1 and R2Bm, the only other model non-LTR retrotransposon system in higher eukaryotes is the Drosophila I-factor, the controlling element in I-R hybrid dysgenesis.22, 23, 24 Chromosomal instability and elevated recombination (both hallmarks of DSB processing) have both been demonstrated in I-R crosses.25, 26, 27
L1 has dramatically impacted the human and mouse genomes through insertional mutagenesis,1 implying that L1 elements are expressed in germline tissues. Insertional mutagenesis is ongoing in the human population as evidenced by de novo and very recent disease-causing insertions.28, 29 In the mouse male germ-line (prospermatogonia), L1 promoters are repressed by the action of the methyltransferase-related Dnmt3L protein.30, 31 While the endogenous amount of L1 expression does not cause infertility in normal individuals, spermatocytes from dnmt3L knockout mice show greatly increased L1 expression. Correlated with this increased expression are aberrant chromosomal structures in meiosis that have been suggested to be due to elevated activity of endogenous retrotransposons and subsequent genetic instability. Though expression of L1 is very low in most normal somatic cells, expression of L1 is elevated in many cancer cells.32, 33, 34 Cancer cells generally show chromosome rearrangements indicative of DSB repair, and DSB repair responses in pre-oncogenic tissues adjacent to cancer cells suggests these lesions contribute to initial tumorigenesis.35, 36 Given the correlations between DSB-related genetic instability, L1 expression, and the predictions of the TPRT model, we sought to characterize the ability of L1 to induce DSBs using immunolocalization of γ-H2AX foci and single-cell gel electrophoresis (COMET) analysis. To further establish the relevance of a DSB intermediate during L1 integration, we tested whether a DSB repair protein, ATM, is important for L1 retrotransposition.
Section snippets
L1 expression induces γ-H2AX foci and fragmented DNA
DSB intermediates, predicted by the TPRT model for L1 insertions (Figure 1), were visualized indirectly by immunofluorescence after expression of a transiently transfected human L1 element, L1.3, which is known to be capable of retrotransposition in HeLa cells. Histone H2AX is phosphorylated in response to ionizing radiation (γ-H2AX) and is detectable as foci in response to DSBs.37, 38 Several quantitative experiments have shown a 1:1 proportion of γ-H2AX foci to the number of IR-induced DSBs,39
Mechanistic implications of L1 DSBs
L1 elements are known to be responsible for a wide range of diseases through insertional mutagenesis.1, 28 The formation of γ-H2AX foci (Figure 2, Figure 5) and COMET assays (Figure 3) demonstrate that expression of the human L1 mobile element creates DSBs as predicted by the TPRT model. The requirement for the endonuclease activity of ORF2 to produce these effects suggests a direct role for ORF2. However, whether the second nick (Figure 1) takes advantage of a separate enzyme for creating
Tissue culture
HeLa cells (ATCC Manassas, VA) were cultured in EMEM plus non-essential amino acids and sodium pyruvate supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco/Invitrogen Carlsbad, CA). YZ-5 and EBS-768 cells were maintained in the same medium but supplemented with hygromycinB at 120 μg/ml to maintain the cATM expression cassette or the corresponding empty vector. The 3T3 and MCF7 (ATCC) cells were cultured in DMEM plus non-essential amino acids and sodium pyruvate supplemented with 10% FBS.
Plasmids
Acknowledgements
We thank Melanie Palmisano for technical help with DNA preparation and tissue culture. We thank Deininger laboratory members, Jeremy Stark, Erik Flemington, and Charles Hemenway for comments on the manuscript. S.L.G. was supported by postdoctoral fellowship PF-01-077-01-LIB from the American Cancer Society and from the Brown Foundation through the Tulane Cancer Center. The P.L.D. laboratory is supported by grants from the USPHS (grant R01GM45668), the National Science Foundation (grant
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