The endonuclease domain of the LINE-1 ORF2 protein can tolerate multiple mutations

Abstract

BackgroundApproximately 17 % of the human genome is comprised of the Long INterspersed Element-1 (LINE-1 or L1) retrotransposon, the only currently active autonomous family of retroelements. Though L1 elements have helped to shape mammalian genome evolution over millions of years, L1 activity can also be mutagenic and result in human disease. L1 expression has the potential to contribute to genomic instability via retrotransposition and DNA double-strand breaks (DSBs). Additionally, L1 is responsible for structural genomic variations induced by other transposable elements such as Alu and SVA, which rely on the L1 ORF2 protein for their propagation. Most of the genomic damage associated with L1 activity originates with the endonuclease domain of the ORF2 protein, which nicks the DNA in preparation for target-primed reverse transcription.ResultsBioinformatic analysis of full-length L1 loci residing in the human genome identified numerous mutations in the amino acid sequence of the ORF2 endonuclease domain. Some of these mutations were found in residues which were predicted to be phosphorylation sites for cellular kinases. We mutated several of these putative phosphorylation sites in the ORF2 endonuclease domain and investigated the effect of these mutations on the function of the full-length ORF2 protein and the endonuclease domain (ENp) alone. Most of the single and multiple point mutations that were tested did not significantly impact expression of the full-length ORF2p, or alter its ability to drive Alu retrotransposition. Similarly, most of those same mutations did not significantly alter expression of ENp, or impair its ability to induce DNA damage and cause toxicity.ConclusionsOverall, our data demonstrate that the full-length ORF2p or the ENp alone can tolerate several specific single and multiple point mutations in the endonuclease domain without significant impairment of their ability to support Alu mobilization or induce DNA damage, respectively.Electronic supplementary materialThe online version of this article (doi:10.1186/s13100-016-0064-x) contains supplementary material, which is available to authorized users.

Cell fitness screens reveal a conflict between LINE-1 retrotransposition and DNA replication

“…5e ). Importantly, LINE-1 cytotoxicity has been previously reported to depend on endonuclease (EN) and reverse transcriptase (RT) activities 8 - 10 , and we confirmed that expression of LINE-1 with inactivating EN and RT mutations is less toxic than wildtype (WT) LINE-1 ( Extended Data Fig. 8 ).…”

“…Many studies have focused on host factors that alter retrotransposition efficiency or on the functional effects of acquired LINE-1 insertions; fewer have focused on cellular effects of LINE-1 expression 6 - 10 . LINE-1 is known to be toxic, but the mechanisms underlying its toxicity are unclear.…”

Involvement of Conserved Amino Acids in the C-Terminal Region of LINE-1 ORF2p in Retrotransposition

“…While we did discover the importance of the Y1180 residue to Alu retrotransposition, it is worth noting that this region of the ORF2p can tolerate individual mutations at other conserved positions. This is similar to the recently described mutagenic tolerance of the ORF2p EN domain (Kines et al 2016). However, there are several important differences between the two.…”

“…To assess the impact of mutation of these amino acids on ORF2p expression, untagged ORF2 constructs with individual amino acids E1165, D1171, Y1180, Y1189 K1190, or Y1232 changed to A were transiently transfected into HeLa cells. Total cell lysate of these cells was subjected to Western blot analysis using ORF2p antibodies specific to amino acids 960-973 (Kines et al 2014;Christian et al 2016a;Kines et al 2016). All constructs expressed their corresponding protein ( Figure 4A).…”

“…All Gal4-tagged constructs ( Figure 2B and Figure 5A) detected using anti-Gal4 DNA binding domain antibodies (sc-577, 1:1000 dilution; Santa Cruz). Full-length ORF2p was detected using previously reported ORF2p antibodies (Kines et al 2014(Kines et al , 2016. Membranes were washed three times in PBS-Tween for 5 min following overnight primary antibody incubation.…”