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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

Expression of ORF2p containing mutations in selected putative phosphorylation sites outside of the endonuclease domain. Top panel: Representative western blot analysis of total cell lysates harvested from HeLa cells transfected with the indicated ORF2 putative phosphorylation mutant constructs. 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 lanes indicate cells transfected with an empty vector. Lysates were probed with polyclonal antibodies generated against the human L1 ORF2 protein. Bottom panel: Western blot quantitation. For each sample, the signal detected for ORF2p was normalized to the total protein load. These relative numbers were expressed as a proportion of the relative number detected from the functional ORF2p. Asterisk denotes a significant difference in the steady-state levels relative to the functional ORF2p (t-test, P ≤ 0.05)
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Fig9: Expression of ORF2p containing mutations in selected putative phosphorylation sites outside of the endonuclease domain. Top panel: Representative western blot analysis of total cell lysates harvested from HeLa cells transfected with the indicated ORF2 putative phosphorylation mutant constructs. 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 lanes indicate cells transfected with an empty vector. Lysates were probed with polyclonal antibodies generated against the human L1 ORF2 protein. Bottom panel: Western blot quantitation. For each sample, the signal detected for ORF2p was normalized to the total protein load. These relative numbers were expressed as a proportion of the relative number detected from the functional ORF2p. Asterisk denotes a significant difference in the steady-state levels relative to the functional ORF2p (t-test, P ≤ 0.05)

Mentions: In contrast to the ORF2 11m protein, ORF2 proteins containing only the S312A, S335A or S312A/S335A mutations supported Alu retrotransposition as efficiently as the functional ORF2p (Fig. 8a). Moreover, there were no significant differences in toxicity after transient expression of these proteins in HeLa and 293 cells (Fig. 8b and c). No statistically significant difference was found between the steady-state expression levels of the functional ORF2p and the ORF2 proteins containing either the individual mutations outside of the endonuclease domain or their combination (Fig. 9).Fig. 8


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)

Expression of ORF2p containing mutations in selected putative phosphorylation sites outside of the endonuclease domain. Top panel: Representative western blot analysis of total cell lysates harvested from HeLa cells transfected with the indicated ORF2 putative phosphorylation mutant constructs. 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 lanes indicate cells transfected with an empty vector. Lysates were probed with polyclonal antibodies generated against the human L1 ORF2 protein. Bottom panel: Western blot quantitation. For each sample, the signal detected for ORF2p was normalized to the total protein load. These relative numbers were expressed as a proportion of the relative number detected from the functional ORF2p. Asterisk denotes a significant difference in the steady-state levels relative to the functional ORF2p (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

Fig9: Expression of ORF2p containing mutations in selected putative phosphorylation sites outside of the endonuclease domain. Top panel: Representative western blot analysis of total cell lysates harvested from HeLa cells transfected with the indicated ORF2 putative phosphorylation mutant constructs. 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 lanes indicate cells transfected with an empty vector. Lysates were probed with polyclonal antibodies generated against the human L1 ORF2 protein. Bottom panel: Western blot quantitation. For each sample, the signal detected for ORF2p was normalized to the total protein load. These relative numbers were expressed as a proportion of the relative number detected from the functional ORF2p. Asterisk denotes a significant difference in the steady-state levels relative to the functional ORF2p (t-test, P ≤ 0.05)
Mentions: In contrast to the ORF2 11m protein, ORF2 proteins containing only the S312A, S335A or S312A/S335A mutations supported Alu retrotransposition as efficiently as the functional ORF2p (Fig. 8a). Moreover, there were no significant differences in toxicity after transient expression of these proteins in HeLa and 293 cells (Fig. 8b and c). No statistically significant difference was found between the steady-state expression levels of the functional ORF2p and the ORF2 proteins containing either the individual mutations outside of the endonuclease domain or their combination (Fig. 9).Fig. 8

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