The human LINE-1 retrotransposon creates DNA double-strand breaks

Abstract

Long interspersed element-1 (L1) is an autonomous retroelement that is active in the human genome. The proposed mechanism of insertion for L1 suggests that cleavage of both strands of genomic DNA is required. We demonstrate that L1 expression leads to a high level of double-strand break (DSB) formation in DNA using immunolocalization of gamma-H2AX foci and the COMET assay. Similar to its role in mediating DSB repair in response to radiation, ATM is required for L1-induced gamma-H2AX foci and for L1 retrotransposition. This is the first characterization of a DNA repair response from expression of a non-long terminal repeat (non-LTR) retrotransposon in mammalian cells as well as the first demonstration that a host DNA repair gene is required for successful integration. Notably, the number of L1-induced DSBs is greater than the predicted numbers of successful insertions, suggesting a significant degree of inefficiency during the integration process. This result suggests that the endonuclease activity of endogenously expressed L1 elements could contribute to DSB formation in germ-line and somatic tissues.

Figure 1

Schematic of the L1 TPRT reaction. The top drawing shows a genomic site with a typical L1 endonuclease cleavage site (5'TTTTAA) that is exposed in the nucleus to the L1 RNP, where the ORF2 cleaves the DNA at the A+T-rich consensus sequence. In the second drawing, the T-rich region primes reverse transcription on the L1 mRNA poly(A) tail. The third drawing highlights the reverse transcriptase activity (formation of the blue cDNA) of the ORF2 and the need for a nick to occur on the second strand, made by an unknown source. The segment between the two nicks is highlighted in gray to illustrate the eventual formation of flanking direct repeats by the duplication of these segments. The fourth panel illustrates the possibility that the second-strand synthesis is primed by microhomology-mediated priming, which results in synthesis of a second copy of the gray segment (dotted arrow). Replication from the gray arrow completes synthesis across the 2nd strand of the cDNA creating a new L1 insert and completing synthesis across the other side of the direct repeat.

Figure 2

L1.3 expression creates DNA DSBs. (a) L1 expression induces the formation of γ-H2AX foci. HeLa cells mock-transfected (control), transfected with the empty expression vector pCEP4 (vector), L1.3 (L1), or L1.3 EN^– (L1 EN^–) over-expressed from pCEP4, were fixed 24 h later, and stained with anti-γ-H2AX antibodies. Untransfected HeLa cells were also treated with 5 Gy of ionizing radiation (IR) and fixed 15 min later as a positive control for focus formation. (b) A time-course on the repair of γ-H2AX from L1 transfected cells in (a) is shown. γ-H2AX immunolocalization in (c) mouse NIH 3T3 cells and in (d) MCF7 cells 24 h after transfection. In addition to expression constructs from (a), an RT mutant, as well as an ORF2 over-expression vector were included in different panels.

Figure 3

COMET assays on L1 transfected cells. HeLa cells were transfected either as a mock (control), or with vector, L1 or L1 EN^– and then subjected to the neutral COMET assay for the periods of time shown.

Figure 4

L1.3 expression induces toxicity and cell cycle arrest. (a) HeLa cells were cotransfected with neoR expression vector and the indicated L1-expression construct or empty vector. Colony formation was assayed after two weeks under neomycin selection. The chart shows the average ±S.E. of four independent experiments. Values are relative to the empty vector control. (b) G2/M arrest induced by L1 expression. HeLa cells were transfected with the noted L1 expression construct or empty vector. At 24 h after transfection, the mitotic index of cells was analyzed using phosphorylated histone 3 staining, shown as % H3-P positive G2 cells in the whole FSC/SSC gated population. The chart shows the mean ± S.E. of three observations of cells from parallel transfections.

Figure 5

L1-induced γ-H2AX foci require ATM. EBS7 (AT–) and YZ-5 (cATM) cells were also analyzed as in Figure 1(a): 10 Gy of IR was used as a positive control.