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

Kines KJ, Sokolowski M, deHaro DL, Christian CM, Baddoo M, Smither ME, Belancio VP - Mob DNA (2016)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Structural and Cellular Biology, Tulane School of Medicine, Tulane Cancer Center and Tulane Center for Aging, New Orleans, LA 70112 USA.

ABSTRACT

Background: Approximately 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.

Results: Bioinformatic 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.

Conclusions: Overall, 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.

No MeSH data available.


Related in: MedlinePlus

Schematic representation of the putative phosphorylation sites within ORF2 mutated in this study. Numbered arrows indicate the locations of the putative phosphorylation sites mutated in this study. The boundaries of the ORF2 endonuclease domain are indicated with green brackets. Plasmids encoding for the full-length ORF2p were generated with the following mutations: S29A; S33A; S37A; S79A; S188A; S228A; S29A/S37A; S79A/T82A; S188A/T189A; S29A/S37A/S228A; S29A/S37A/S188A/T189A; S312A; S335A; and S312A/S335A. ORF2 11m contains the following mutations (red ovals): S29A/S33A/S37A/S151A/S188A/T189A/T220A/T224A/S228A/S312A/S335A. Plasmids encoding for the endonuclease domain (ENp) alone were generated with the following mutations: S29A; S33A; S37A; S79A; S188A; S228A; S29A/S37A; S79A/T82A; S188A/T189A; S29A/S37A/S228A; and S29A/S37A/S188A/T189A. EN 9m contains the following mutations (blue boxes): S29A/S33A/S37A/S151A/S188A/T189A/T220A/T224A/S228A. Plasmids encoding for the full-length L1 containing the following mutations within ORF2 were generated: S29A; S33A; S312A; S335A; and S312A/S335A
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Fig2: Schematic representation of the putative phosphorylation sites within ORF2 mutated in this study. Numbered arrows indicate the locations of the putative phosphorylation sites mutated in this study. The boundaries of the ORF2 endonuclease domain are indicated with green brackets. Plasmids encoding for the full-length ORF2p were generated with the following mutations: S29A; S33A; S37A; S79A; S188A; S228A; S29A/S37A; S79A/T82A; S188A/T189A; S29A/S37A/S228A; S29A/S37A/S188A/T189A; S312A; S335A; and S312A/S335A. ORF2 11m contains the following mutations (red ovals): S29A/S33A/S37A/S151A/S188A/T189A/T220A/T224A/S228A/S312A/S335A. Plasmids encoding for the endonuclease domain (ENp) alone were generated with the following mutations: S29A; S33A; S37A; S79A; S188A; S228A; S29A/S37A; S79A/T82A; S188A/T189A; S29A/S37A/S228A; and S29A/S37A/S188A/T189A. EN 9m contains the following mutations (blue boxes): S29A/S33A/S37A/S151A/S188A/T189A/T220A/T224A/S228A. Plasmids encoding for the full-length L1 containing the following mutations within ORF2 were generated: S29A; S33A; S312A; S335A; and S312A/S335A

Mentions: As our main interest is in understanding the impact of mutations on L1 endonuclease function, we chose to investigate the functional impact of mutations in putative phosphorylation sites because, if found relevant, these sites could also play a regulatory role. Amino acid residues that were scored by both the ELM and NetPhos prediction programs were selected for mutagenesis (Additional file 3: Table S2). We generated expression plasmids containing codon-optimized human L1 ORF2 sequence with either serine to alanine (S to A) or threonine to alanine (T to A) point mutations in putative phosphorylation sites (Fig. 2). The resulting plasmids contained one (S29A; S33A; S37A; S79A; S188A; S228A), two (S29A/S37A; S79A/T82A; S188A/T189A), three (S29A/S37A/S228A), or four (S29A/S37A/S188A/T189A) point mutations in putative phosphorylation sites within the endonuclease domain. The ORF2 11m construct was designed to include mutations in sites predicted to be phosphorylated by kinases in the CMGC group (CDK, MAPK, GSK3 and CLK) [40]. ORF2 11m contains 11 mutations (S29A/S33A/S37A/S151A/S188A/T189A/T220A/T224A/S228A/S312A/S335A); 9 mutations are in the putative phosphorylation sites within the endonuclease domain and the remaining 2 are located between the endonuclease and z-motif region. Because these mutant constructs were tested in transiently transfected HeLa and 293 cells, we used an NGS RNAseq approach to confirm that these cell lines express many cellular kinases (Additional file 5: Table S3) [41].Fig. 2


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

Kines KJ, Sokolowski M, deHaro DL, Christian CM, Baddoo M, Smither ME, Belancio VP - Mob DNA (2016)

Schematic representation of the putative phosphorylation sites within ORF2 mutated in this study. Numbered arrows indicate the locations of the putative phosphorylation sites mutated in this study. The boundaries of the ORF2 endonuclease domain are indicated with green brackets. Plasmids encoding for the full-length ORF2p were generated with the following mutations: S29A; S33A; S37A; S79A; S188A; S228A; S29A/S37A; S79A/T82A; S188A/T189A; S29A/S37A/S228A; S29A/S37A/S188A/T189A; S312A; S335A; and S312A/S335A. ORF2 11m contains the following mutations (red ovals): S29A/S33A/S37A/S151A/S188A/T189A/T220A/T224A/S228A/S312A/S335A. Plasmids encoding for the endonuclease domain (ENp) alone were generated with the following mutations: S29A; S33A; S37A; S79A; S188A; S228A; S29A/S37A; S79A/T82A; S188A/T189A; S29A/S37A/S228A; and S29A/S37A/S188A/T189A. EN 9m contains the following mutations (blue boxes): S29A/S33A/S37A/S151A/S188A/T189A/T220A/T224A/S228A. Plasmids encoding for the full-length L1 containing the following mutations within ORF2 were generated: S29A; S33A; S312A; S335A; and S312A/S335A
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Fig2: Schematic representation of the putative phosphorylation sites within ORF2 mutated in this study. Numbered arrows indicate the locations of the putative phosphorylation sites mutated in this study. The boundaries of the ORF2 endonuclease domain are indicated with green brackets. Plasmids encoding for the full-length ORF2p were generated with the following mutations: S29A; S33A; S37A; S79A; S188A; S228A; S29A/S37A; S79A/T82A; S188A/T189A; S29A/S37A/S228A; S29A/S37A/S188A/T189A; S312A; S335A; and S312A/S335A. ORF2 11m contains the following mutations (red ovals): S29A/S33A/S37A/S151A/S188A/T189A/T220A/T224A/S228A/S312A/S335A. Plasmids encoding for the endonuclease domain (ENp) alone were generated with the following mutations: S29A; S33A; S37A; S79A; S188A; S228A; S29A/S37A; S79A/T82A; S188A/T189A; S29A/S37A/S228A; and S29A/S37A/S188A/T189A. EN 9m contains the following mutations (blue boxes): S29A/S33A/S37A/S151A/S188A/T189A/T220A/T224A/S228A. Plasmids encoding for the full-length L1 containing the following mutations within ORF2 were generated: S29A; S33A; S312A; S335A; and S312A/S335A
Mentions: As our main interest is in understanding the impact of mutations on L1 endonuclease function, we chose to investigate the functional impact of mutations in putative phosphorylation sites because, if found relevant, these sites could also play a regulatory role. Amino acid residues that were scored by both the ELM and NetPhos prediction programs were selected for mutagenesis (Additional file 3: Table S2). We generated expression plasmids containing codon-optimized human L1 ORF2 sequence with either serine to alanine (S to A) or threonine to alanine (T to A) point mutations in putative phosphorylation sites (Fig. 2). The resulting plasmids contained one (S29A; S33A; S37A; S79A; S188A; S228A), two (S29A/S37A; S79A/T82A; S188A/T189A), three (S29A/S37A/S228A), or four (S29A/S37A/S188A/T189A) point mutations in putative phosphorylation sites within the endonuclease domain. The ORF2 11m construct was designed to include mutations in sites predicted to be phosphorylated by kinases in the CMGC group (CDK, MAPK, GSK3 and CLK) [40]. ORF2 11m contains 11 mutations (S29A/S33A/S37A/S151A/S188A/T189A/T220A/T224A/S228A/S312A/S335A); 9 mutations are in the putative phosphorylation sites within the endonuclease domain and the remaining 2 are located between the endonuclease and z-motif region. Because these mutant constructs were tested in transiently transfected HeLa and 293 cells, we used an NGS RNAseq approach to confirm that these cell lines express many cellular kinases (Additional file 5: Table S3) [41].Fig. 2

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Structural and Cellular Biology, Tulane School of Medicine, Tulane Cancer Center and Tulane Center for Aging, New Orleans, LA 70112 USA.

ABSTRACT

Background: Approximately 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.

Results: Bioinformatic 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.

Conclusions: Overall, 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.

No MeSH data available.


Related in: MedlinePlus