Limits...
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 and detection of ORF2 proteins containing mutations in putative phosphorylation sites. 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 RT- is a non-functional protein containing a mutation in the reverse transcriptase (D702A) domain. 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
getmorefigures.php?uid=PMC4837594&req=5

Fig5: Expression and detection of ORF2 proteins containing mutations in putative phosphorylation sites. 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 RT- is a non-functional protein containing a mutation in the reverse transcriptase (D702A) domain. 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: To determine whether the significant reduction in Alu retrotransposition driven by the ORF2 11m mutant was a result of altered protein expression, we analyzed total protein lysates harvested from HeLa cells transiently transfected with each of the ORF2 mutant plasmids described above. Western blot analysis using antibodies specific to the human L1 ORF2 protein [42, 43] detected ORF2p in the total lysates of transfected HeLa cells (Fig. 5). Quantitation of the relative ORF2p expression levels revealed an approximately 50 % reduction in the steady-state levels of the ORF2 11m protein in comparison to the functional ORF2p. No statistically significant differences in expression were observed between the functional ORF2p and any of the other ORF2 proteins containing mutations in putative phosphorylation sites. The same lysates were also probed with anti-γH2AX antibodies, as histone H2AX is phosphorylated in response to DNA DSBs and can therefore be used as an indication of DNA damage (Additional file 7: Figure S4) [34]. Consistent with our toxicity results in HeLa cells, expression of the functional ORF2p or any of the ORF2 putative phosphorylation mutant proteins generated γH2AX signals that were not significantly different than the background signal observed with the empty vector control or non-functional ORF2 protein (t-test, P ≥ 0.05).Fig. 5


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 and detection of ORF2 proteins containing mutations in putative phosphorylation sites. 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 RT- is a non-functional protein containing a mutation in the reverse transcriptase (D702A) domain. 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

Fig5: Expression and detection of ORF2 proteins containing mutations in putative phosphorylation sites. 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 RT- is a non-functional protein containing a mutation in the reverse transcriptase (D702A) domain. 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: To determine whether the significant reduction in Alu retrotransposition driven by the ORF2 11m mutant was a result of altered protein expression, we analyzed total protein lysates harvested from HeLa cells transiently transfected with each of the ORF2 mutant plasmids described above. Western blot analysis using antibodies specific to the human L1 ORF2 protein [42, 43] detected ORF2p in the total lysates of transfected HeLa cells (Fig. 5). Quantitation of the relative ORF2p expression levels revealed an approximately 50 % reduction in the steady-state levels of the ORF2 11m protein in comparison to the functional ORF2p. No statistically significant differences in expression were observed between the functional ORF2p and any of the other ORF2 proteins containing mutations in putative phosphorylation sites. The same lysates were also probed with anti-γH2AX antibodies, as histone H2AX is phosphorylated in response to DNA DSBs and can therefore be used as an indication of DNA damage (Additional file 7: Figure S4) [34]. Consistent with our toxicity results in HeLa cells, expression of the functional ORF2p or any of the ORF2 putative phosphorylation mutant proteins generated γH2AX signals that were not significantly different than the background signal observed with the empty vector control or non-functional ORF2 protein (t-test, P ≥ 0.05).Fig. 5

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