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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

Acute toxicity assay in HeLa and 293 cells transiently transfected with ORF2 putative phosphorylation mutant plasmids. a HeLa cells were cotransfected with a NeoR expression vector and the indicated ORF2 putative phosphorylation mutant plasmid. b 293 cells were cotransfected with a NeoR expression vector and the indicated ORF2 putative phosphorylation mutant plasmid. In both panel a and b ORF2 is the functional protein and ORF2 EN-RT- is a non-functional protein containing mutations in the endonuclease (D205A) and reverse transcriptase (D702A) domains. Control indicates cells transfected with an empty vector and the NeoR expression vector. Colony formation was assayed after 2 weeks under G418 selection (Y-axis) and used as a measure of toxicity as previously described [26, 42]
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Fig4: Acute toxicity assay in HeLa and 293 cells transiently transfected with ORF2 putative phosphorylation mutant plasmids. a HeLa cells were cotransfected with a NeoR expression vector and the indicated ORF2 putative phosphorylation mutant plasmid. b 293 cells were cotransfected with a NeoR expression vector and the indicated ORF2 putative phosphorylation mutant plasmid. In both panel a and b ORF2 is the functional protein and ORF2 EN-RT- is a non-functional protein containing mutations in the endonuclease (D205A) and reverse transcriptase (D702A) domains. Control indicates cells transfected with an empty vector and the NeoR expression vector. Colony formation was assayed after 2 weeks under G418 selection (Y-axis) and used as a measure of toxicity as previously described [26, 42]

Mentions: In addition to genomic damage due to retrotransposition, expression of the ORF2 protein can cause cellular toxicity in a dose-dependent manner when ectopically expressed at high levels [26, 27]. A positive or negative change in ORF2p toxicity may mask or cause subsequent variations in ORF2p-driven Alu mobilization. Using a previously described assay [26], we measured acute toxicity following transient transfection of the ORF2 putative phosphorylation plasmids to determine if variations in cellular toxicity may contribute to the observed reduction in Alu retrotransposition driven by the ORF2 11m protein (Additional file 6: Figure S3A). For this reason, the same amount of DNA that was transfected in the retrotransposition assay was used for evaluation of potential changes in ORF2p toxicity. Results in Fig. 4a demonstrate that there were no significant differences in toxicity between the functional ORF2p and any of the putative phosphorylation mutant proteins after transient expression in HeLa cells. In contrast to previously reported results [26], we did not observe any toxicity associated with the expression of functional or mutant ORF2 proteins in HeLa cells under our experimental conditions. This discrepancy is likely due to the 20-fold difference in the amount of plasmid DNA transfected per cell between the reported transfection conditions (2 μg plasmid per 100,000 cells in a 6-well plate) and the conditions used here (0.5 μg per 500,000 cells in a T75 flask). Additionally, it was reported that expression of the full-length ORF2p alone was not as efficient in generating γH2AX foci in HeLa cells as was the expression of the full-length L1 [26]. We did observe toxicity after transient expression of the functional ORF2p and putative phosphorylation mutant proteins in 293 cells (Fig. 4b). Consistent with the results obtained in HeLa cells, no significant differences between the toxicity observed after expression of the functional and mutated ORF2 proteins were detected.Fig. 4


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)

Acute toxicity assay in HeLa and 293 cells transiently transfected with ORF2 putative phosphorylation mutant plasmids. a HeLa cells were cotransfected with a NeoR expression vector and the indicated ORF2 putative phosphorylation mutant plasmid. b 293 cells were cotransfected with a NeoR expression vector and the indicated ORF2 putative phosphorylation mutant plasmid. In both panel a and b ORF2 is the functional protein and ORF2 EN-RT- is a non-functional protein containing mutations in the endonuclease (D205A) and reverse transcriptase (D702A) domains. Control indicates cells transfected with an empty vector and the NeoR expression vector. Colony formation was assayed after 2 weeks under G418 selection (Y-axis) and used as a measure of toxicity as previously described [26, 42]
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig4: Acute toxicity assay in HeLa and 293 cells transiently transfected with ORF2 putative phosphorylation mutant plasmids. a HeLa cells were cotransfected with a NeoR expression vector and the indicated ORF2 putative phosphorylation mutant plasmid. b 293 cells were cotransfected with a NeoR expression vector and the indicated ORF2 putative phosphorylation mutant plasmid. In both panel a and b ORF2 is the functional protein and ORF2 EN-RT- is a non-functional protein containing mutations in the endonuclease (D205A) and reverse transcriptase (D702A) domains. Control indicates cells transfected with an empty vector and the NeoR expression vector. Colony formation was assayed after 2 weeks under G418 selection (Y-axis) and used as a measure of toxicity as previously described [26, 42]
Mentions: In addition to genomic damage due to retrotransposition, expression of the ORF2 protein can cause cellular toxicity in a dose-dependent manner when ectopically expressed at high levels [26, 27]. A positive or negative change in ORF2p toxicity may mask or cause subsequent variations in ORF2p-driven Alu mobilization. Using a previously described assay [26], we measured acute toxicity following transient transfection of the ORF2 putative phosphorylation plasmids to determine if variations in cellular toxicity may contribute to the observed reduction in Alu retrotransposition driven by the ORF2 11m protein (Additional file 6: Figure S3A). For this reason, the same amount of DNA that was transfected in the retrotransposition assay was used for evaluation of potential changes in ORF2p toxicity. Results in Fig. 4a demonstrate that there were no significant differences in toxicity between the functional ORF2p and any of the putative phosphorylation mutant proteins after transient expression in HeLa cells. In contrast to previously reported results [26], we did not observe any toxicity associated with the expression of functional or mutant ORF2 proteins in HeLa cells under our experimental conditions. This discrepancy is likely due to the 20-fold difference in the amount of plasmid DNA transfected per cell between the reported transfection conditions (2 μg plasmid per 100,000 cells in a 6-well plate) and the conditions used here (0.5 μg per 500,000 cells in a T75 flask). Additionally, it was reported that expression of the full-length ORF2p alone was not as efficient in generating γH2AX foci in HeLa cells as was the expression of the full-length L1 [26]. We did observe toxicity after transient expression of the functional ORF2p and putative phosphorylation mutant proteins in 293 cells (Fig. 4b). Consistent with the results obtained in HeLa cells, no significant differences between the toxicity observed after expression of the functional and mutated ORF2 proteins were detected.Fig. 4

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