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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

Alu retrotransposition driven by ORF2 proteins containing mutations in putative phosphorylation sites. ORF2 proteins containing mutations in the indicated putative phosphorylation sites were used to drive Alu retrotransposition in HeLa cells, as previously described [3]. 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 Alu retrotransposition reporter plasmid. The graph depicts the relative number of Alu retrotransposition events as represented by NeoR colonies (Y-axis). Asterisks indicate a statistically significant difference in Alu retrotransposition compared to ORF2 (t-test, P ≤ 0.05)
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Fig3: Alu retrotransposition driven by ORF2 proteins containing mutations in putative phosphorylation sites. ORF2 proteins containing mutations in the indicated putative phosphorylation sites were used to drive Alu retrotransposition in HeLa cells, as previously described [3]. 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 Alu retrotransposition reporter plasmid. The graph depicts the relative number of Alu retrotransposition events as represented by NeoR colonies (Y-axis). Asterisks indicate a statistically significant difference in Alu retrotransposition compared to ORF2 (t-test, P ≤ 0.05)

Mentions: As an indication of protein function, we investigated the ability of the ORF2 proteins containing the above described mutations to mobilize Alu using a previously described retrotransposition assay [3]. With the exception of the ORF2 11m mutant, all of the full-length mutant ORF2 proteins supported Alu retrotransposition at similar levels to the functional ORF2p (t-test, P ≥ 0.05) when transiently expressed in HeLa cells (Fig. 3). A significant ~50 % decrease in retrotransposition was observed when Alu mobilization was driven by the ORF2 11m mutant protein (t-test, P ≤ 0.05).Fig. 3


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)

Alu retrotransposition driven by ORF2 proteins containing mutations in putative phosphorylation sites. ORF2 proteins containing mutations in the indicated putative phosphorylation sites were used to drive Alu retrotransposition in HeLa cells, as previously described [3]. 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 Alu retrotransposition reporter plasmid. The graph depicts the relative number of Alu retrotransposition events as represented by NeoR colonies (Y-axis). Asterisks indicate a statistically significant difference in Alu retrotransposition compared to ORF2 (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

Fig3: Alu retrotransposition driven by ORF2 proteins containing mutations in putative phosphorylation sites. ORF2 proteins containing mutations in the indicated putative phosphorylation sites were used to drive Alu retrotransposition in HeLa cells, as previously described [3]. 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 Alu retrotransposition reporter plasmid. The graph depicts the relative number of Alu retrotransposition events as represented by NeoR colonies (Y-axis). Asterisks indicate a statistically significant difference in Alu retrotransposition compared to ORF2 (t-test, P ≤ 0.05)
Mentions: As an indication of protein function, we investigated the ability of the ORF2 proteins containing the above described mutations to mobilize Alu using a previously described retrotransposition assay [3]. With the exception of the ORF2 11m mutant, all of the full-length mutant ORF2 proteins supported Alu retrotransposition at similar levels to the functional ORF2p (t-test, P ≥ 0.05) when transiently expressed in HeLa cells (Fig. 3). A significant ~50 % decrease in retrotransposition was observed when Alu mobilization was driven by the ORF2 11m mutant protein (t-test, P ≤ 0.05).Fig. 3

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