Determinants for DNA target structure selectivity of the human LINE-1 retrotransposon endonuclease

The human LINE-1 endonuclease (L1-EN) is the targeting endonuclease encoded by the human LINE-1 (L1) retrotransposon. L1-EN guides the genomic integration of new L1 and Alu elements that presently account for ~28% of the human genome. L1-EN bears considerable technological interest, because its target selectivity may ultimately be engineered to allow the site-specific integration of DNA into defined genomic locations. Based on the crystal structure, we generated L1-EN mutants to analyze and manipulate DNA target site recognition. Crystal structures and their dynamic and functional analysis show entire loop grafts to be feasible, resulting in altered specificity, while individual point mutations do not change the nicking pattern of L1-EN. Structural parameters of the DNA target seem more important for recognition than the nucleotide sequence, and nicking profiles on DNA oligonucleotides in vitro are less well defined than the respective integration site consensus in vivo. This suggests that additional factors other than the DNA nicking specificity of L1-EN contribute to the targeted integration of non-LTR retrotransposons.     

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

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.     

Insights into the DNA cleavage mechanism of human LINE-1 retrotransposon endonuclease

The human LINE-1 endonuclease (L1-EN) contributes in defining the genomic integration sites of the abundant human L1 and Alu retrotransposons. LINEs have been considered as possible vehicles for gene delivery and understanding the mechanism of L1-EN could help engineering them as genetic tools. We tested the in vitro activity of point mutants in three L1-EN residues--Asp145, Arg155, Ile204--that are key for DNA cleavage, and determined their crystal structures. The L1-EN structure remains overall unaffected by the mutations, which change the enzyme activity but leave DNA cleavage sequence specificity mostly unaffected. To better understand the mechanism of L1-EN, we performed molecular dynamics simulations using as model the structures of wild type EN-L1, of two betaB6-betaB5 loop exchange mutants we have described previously to be important for DNA recognition, of the R155A mutant from this study, and of the homologous TRAS1 endonuclease: all confirm a rigid scaffold. The simulations crucially indicate that the betaB6-betaB5 loop shows an anticorrelated motion with the surface loops betaA6-betaA5 and betaB3-alphaB1. The latter loop harbors N118, a residue that alters DNA cleavage specificity in homologous endonucleases, and implies that the plasticity and correlated motion of these loops has a functional importance in DNA recognition and binding. To further explore how these loops are possibly involved in DNA binding, we docked computationally two DNA substrates to our structure, one involving a flipped-out nucleotide downstream the scissile phosphodiester; and one not. The models for both scenarios are feasible and agree with the hypotheses derived from the dynamic simulations. The reduced cleavage activity we have observed for the I204Y mutant above however, favors the flipped out nucleotide model.     

NeSL-1, an ancient lineage of site-specific non-LTR retrotransposons from Caenorhabditis elegans

Phylogenetic analyses of non-LTR retrotransposons suggest that all elements can be divided into 11 lineages. The 3 oldest lineages show target site specificity for unique locations in the genome and encode an endonuclease with an active site similar to certain restriction enzymes. The more "modern" non-LTR lineages possess an apurinic endonuclease-like domain and generally lack site specificity. The genome sequence of Caenorhabditis elegans reveals the presence of a non-LTR retrotransposon that resembles the older elements, in that it contains a single open reading frame with a carboxyl-terminal restriction-like endonuclease domain. Located near the N-terminal end of the ORF is a cysteine protease domain not found in any other non-LTR element. The N2 strain of C. elegans appears to contain only one full-length and several 5' truncated copies of this element. The elements specifically insert in the Spliced leader-1 genes; hence the element has been named NeSL-1 (Nematode Spliced Leader-1). Phylogenetic analysis confirms that NeSL-1 branches very early in the non-LTR lineage and that it represents a 12th lineage of non-LTR elements. The target specificity of NeSL-1 for the spliced leader exons and the similarity of its structure to that of R2 elements leads to a simple model for its expression and retrotransposition.     

Comparative studies of the endonucleases from two related Xenopus laevis retrotransposons, Tx1L and Tx2L: target site specificity and evolutionary implications

In the genome of the South African frog, Xenopus laevis, there are two complex families of transposable elements, Tx1 and Tx2, that have identical overall structures, but distinct sequences. In each family there are approximately 1500 copies of an apparent DNA-based element (Tx1D and Tx2D). Roughly 10% of these elements in each family are interrupted by a non-LTR retrotransposon (Tx1L and Tx2L). Each retrotransposon is flanked by a 23-bp target duplication of a specific D element sequence. In earlier work, we showed that the endonuclease domain (Tx1L EN) located in the second open reading frame (ORF2) of Tx1L encodes a protein that makes a single-strand cut precisely at the expected site within its target sequence, supporting the idea that Tx1L is a site-specific retrotransposon. In this study, we express the endonuclease domain of Tx2L (Tx2L EN) and compare the target preferences of the two enzymes. Each endonuclease shows some preference for its cognate target, on the order of 5-fold over the non- cognate target. The observed discrimination is not sufficient, however, to explain the observation that no cross-occupancy is observed – that is, L elements of one family have never been found within D elements of the other family. Possible sources of additional specificity are discussed. We also compare two hypotheses regarding the genome duplication event that led to the contemporary pseudotetraploid character of Xenopus laevis in light of the Tx1L and Tx2L data. This revised version was published online in July 2006 with corrections to the Cover Date.     

Selective inhibition of Alu retrotransposition by APOBEC3G

The non-LTR retrotransposon LINE-1 (L1) comprises  17% of the human genome, and the L1-encoded proteins can function in trans to mediate the retrotransposition of non-autonomous retrotransposons (i.e., Alu and probably SVA elements) and cellular mRNAs to generate processed pseudogenes. Here, we have examined the effect of APOBEC3G and APOBEC3F, cytidine deaminases that inhibit Vif-deficient HIV-1 replication, on Alu retrotransposition and other L1-mediated retrotransposition processes. We demonstrate that APOBEC3G selectively inhibits Alu retrotransposition in an ORF1p-independent manner. An active cytidine deaminase site is not required for the inhibition of Alu retrotransposition and the resultant integration events lack G to A or C to T hypermutation. These data demonstrate a differential restriction of L1 and Alu retrotransposition by APOBEC3G, and suggest that the Alu ribonucleoprotein complex may be targeted by APOBEC3G.     

A non-LTR retroelement extinction in Spermophilus tridecemlineatus

The typical mammalian genome is dominated by two types of transposable elements (TEs), the autonomous and non-autonomous non-LTR retrotransposons, i.e. LINEs and SINEs, and with few exceptions there is a sole active LINE family (L1). During an ongoing investigation of TEs in rodents we determined that overall transposon activity has been steadily declining in Spermophilus tridecemlineatus. More specifically, the typically ubiquitous L1 activity of mammals has decreased drastically within the last 26MY. Indeed, only three L1 insertions with intact ORF1 sequences were readily identifiable and no intact ORF2 sequences were identified. The last L1 and SINE insertions date to ~5.3MYA and 4MYA, respectively. Based on our inability to computationally identify recently inserted L1 elements we suggest that S. tridecemlineatus is experiencing a quiescence or extinction of non-LTR retrotransposon activity. Such a finding represents only the fourth instance of a loss of non-LTR retrotransposon activity identified in mammals and, as such, represents an important additional data point to guide our understanding of LINE dynamics in eutherians.