Potential for genomic instability associated with retrotranspositionally-incompetent L1 loci

Abstract

Expression of the L1 retrotransposon can damage the genome through insertional mutagenesis and the generation of DNA double-strand breaks (DSBs). The majority of L1 loci in the human genome are 5′-truncated and therefore incapable of retrotransposition. While thousands of full-length L1 loci remain, most are retrotranspositionally-incompetent due to inactivating mutations. However, mutations leading to premature stop codons within the L1 ORF2 sequence may yield truncated proteins that retain a functional endonuclease domain. We demonstrate that some truncated ORF2 proteins cause varying levels of toxicity and DNA damage when chronically overexpressed in mammalian cells. Furthermore, transfection of some ORF2 constructs containing premature stop codons supported low levels of Alu retrotransposition, demonstrating the potential for select retrotranspositionally-incompetent L1 loci to generate genomic instability. This result suggests yet another plausible explanation for the relative success of Alu elements in populating the human genome. Our data suggest that a subset of retrotranspositionally-incompetent L1s, previously considered to be harmless to genomic integrity, may have the potential to cause chronic DNA damage by introducing DSBs and mobilizing Alu. These results imply that the number of known L1 loci in the human genome that potentially threaten its stability may not be limited to the retrotranspositionally active loci.

Development of a monoclonal antibody specific to the endonuclease domain of the human LINE-1 ORF2 protein

“…This approach determined that the anti-ORF2p monoclonal antibody does not detect mouse ORF2 or EN proteins (mORF2p and mENp, respectively) even though it detected both human ENp and ORF2p (Figure 2A, monoclonal Ab panel). The mouse ENp and ORF2p were detected when Western blot analysis was performed with polyclonal antibodies raised against the endonuclease domain of the mouse ORF2p [28] (Figure 2B, mouse Ab panel) confirming that the proteins are expressed under these transfection conditions. Anti-ORF2p monoclonal antibody recognizes an epitope which includes amino acid 205 of the human ORF2p endonuclease…”

“…This approach demonstrated that the anti-ORF2p monoclonal antibody detects ENp containing the H230A mutation, but not the ENp with the D205A mutation ( Figure 3B, monoclonal). Both ENp mutants are readily detected with the antiORF2p polyclonal antibody [27,28], demonstrating that both proteins are produced under these transfection conditions ( Figure 3B, polyclonal). A similar result was obtained when the monoclonal anti-ORF2p antibody was used to detect transiently expressed functional (ORF2) and non-functional (single and double mutants) fulllength human ORF2 proteins (ORF2 205, ORF2 230, and ORF2 205,230, respectively) as well as truncated, functional, and double mutant human ORF2 proteins (ENz and ENRT) [28] (Figure 4A-E, ORF2, ENz, and ENRT).…”

“…Both ENp mutants are readily detected with the antiORF2p polyclonal antibody [27,28], demonstrating that both proteins are produced under these transfection conditions ( Figure 3B, polyclonal). A similar result was obtained when the monoclonal anti-ORF2p antibody was used to detect transiently expressed functional (ORF2) and non-functional (single and double mutants) fulllength human ORF2 proteins (ORF2 205, ORF2 230, and ORF2 205,230, respectively) as well as truncated, functional, and double mutant human ORF2 proteins (ENz and ENRT) [28] (Figure 4A-E, ORF2, ENz, and ENRT). The anti-ORF2p monoclonal antibody specifically detected functional, but not the non-functional ENz, ENRT, and ORF2 proteins containing the D205A and H230A mutations, even though all of these proteins were produced in these cells as confirmed by Western blot analysis using polyclonal anti-ORF2p antibodies ( Figure 4D,E).…”

“…Western blot analysis of total cellular lysates from human and mouse cells transiently transfected with EN or EN 205, 230 plasmids containing codon-optimized sequences producing functional or non-functional (D205A, H230A double mutant) human endonucleases demonstrated that the anti-ORF2p monoclonal antibody detects the active, but not the mutated, endonuclease protein (Figure 3A, monoclonal; Additional file 1: Figure S1, monoclonal). Both proteins were detected using the polyclonal anti-ORF2p antibody [28] (Figure 3A; Additional file 1: Figure S1). …”

“…Human and mouse L1 ORF2 proteins exhibit a high degree of sequence homology and conservation of function making findings in mouse model systems biologically relevant to the replication cycle of the human L1 [25,26]. Although much has been learned about ORF2p function in vitro and in mammalian cells using overexpressed tagged ORF2 proteins and polyclonal antiORF2p antibodies [27][28][29][30], having a monoclonal antibody that can detect the untagged human ORF2 protein would be a useful molecular tool to study the requirements for the human L1 ORF2p expression and activity. It would also aid in advancing our appreciation of the ORF2p impact on host genome stability and in understanding the consequences of its activity to human health.…”