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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

Chronic expression of EN proteins containing mutations in putative phosphorylation sites causes toxicity. HeLa cells were transfected with a single expression plasmid containing both HygroR and the indicated EN putative phosphorylation mutant sequence. Hygromycin selection was maintained for 2 weeks post-transfection, allowing stable expression of ENp throughout the assay. EN is the functional protein and EN- is a non-functional protein containing inactivating mutations (D205A/H230A). Control indicates cells transfected with an empty vector. Colony formation was assayed after 2 weeks under hygromycin (Y-axis) and used as a measure of toxicity as previously described [27, 43]. Asterisks indicate a statistically significant difference in the relative number of HygroR colonies compared to EN (t-test, P ≤ 0.05)
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Fig6: Chronic expression of EN proteins containing mutations in putative phosphorylation sites causes toxicity. HeLa cells were transfected with a single expression plasmid containing both HygroR and the indicated EN putative phosphorylation mutant sequence. Hygromycin selection was maintained for 2 weeks post-transfection, allowing stable expression of ENp throughout the assay. EN is the functional protein and EN- is a non-functional protein containing inactivating mutations (D205A/H230A). Control indicates cells transfected with an empty vector. Colony formation was assayed after 2 weeks under hygromycin (Y-axis) and used as a measure of toxicity as previously described [27, 43]. Asterisks indicate a statistically significant difference in the relative number of HygroR colonies compared to EN (t-test, P ≤ 0.05)

Mentions: Using a previously reported assay [27, 43], we measured chronic toxicity of these constructs in HeLa cells (Additional file 6: Figure S3B; Fig. 6). With the exception of the EN 9m mutant, all of the mutant endonuclease proteins were as toxic as the functional ENp. Chronic expression of the EN 9m mutant protein resulted in a statistically significant 2.5-fold difference in the relative colony number in comparison to the functional ENp (Fig. 6). Similar results were obtained after transient transfection of HeLa cells with the EN mutant plasmids in an acute toxicity assay (Additional file 6: Figure S3A; Additional file 8: Figure S5).Fig. 6


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)

Chronic expression of EN proteins containing mutations in putative phosphorylation sites causes toxicity. HeLa cells were transfected with a single expression plasmid containing both HygroR and the indicated EN putative phosphorylation mutant sequence. Hygromycin selection was maintained for 2 weeks post-transfection, allowing stable expression of ENp throughout the assay. EN is the functional protein and EN- is a non-functional protein containing inactivating mutations (D205A/H230A). Control indicates cells transfected with an empty vector. Colony formation was assayed after 2 weeks under hygromycin (Y-axis) and used as a measure of toxicity as previously described [27, 43]. Asterisks indicate a statistically significant difference in the relative number of HygroR colonies compared to EN (t-test, P ≤ 0.05)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4837594&req=5

Fig6: Chronic expression of EN proteins containing mutations in putative phosphorylation sites causes toxicity. HeLa cells were transfected with a single expression plasmid containing both HygroR and the indicated EN putative phosphorylation mutant sequence. Hygromycin selection was maintained for 2 weeks post-transfection, allowing stable expression of ENp throughout the assay. EN is the functional protein and EN- is a non-functional protein containing inactivating mutations (D205A/H230A). Control indicates cells transfected with an empty vector. Colony formation was assayed after 2 weeks under hygromycin (Y-axis) and used as a measure of toxicity as previously described [27, 43]. Asterisks indicate a statistically significant difference in the relative number of HygroR colonies compared to EN (t-test, P ≤ 0.05)
Mentions: Using a previously reported assay [27, 43], we measured chronic toxicity of these constructs in HeLa cells (Additional file 6: Figure S3B; Fig. 6). With the exception of the EN 9m mutant, all of the mutant endonuclease proteins were as toxic as the functional ENp. Chronic expression of the EN 9m mutant protein resulted in a statistically significant 2.5-fold difference in the relative colony number in comparison to the functional ENp (Fig. 6). Similar results were obtained after transient transfection of HeLa cells with the EN mutant plasmids in an acute toxicity assay (Additional file 6: Figure S3A; Additional file 8: Figure S5).Fig. 6

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