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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 EN proteins containing mutations in putative phosphorylation sites can induce DNA damage. a Representative western blot analysis of total cell lysates harvested from HeLa cells transiently transfected with the indicated EN putative phosphorylation mutant plasmids. EN is the functional protein and EN- is a non-functional protein containing inactivating mutations (D205A/H230A). Control lanes indicate cells transfected with an empty vector. a Lysates were probed with polyclonal antibodies generated against the human L1 ORF2 endonuclease domain [42, 43], top panel; anti-γH2AX antibodies to detect the phosphorylation of histone H2AX in response to DNA damage, middle panel; and anti-GAPDH to serve as a loading control, bottom panel. b Western blot quantitation. For each sample, the signal detected for ENp was normalized to the signal detected for GAPDH. These relative numbers were expressed as a proportion of the relative number detected from the functional ENp. Asterisk denotes a significant difference in the steady-state levels relative to the functional ENp (t-test, P ≤ 0.05). c Western blot quantitation. For each sample, the signal detected for γH2AX was normalized to the signal detected for GAPDH. These relative numbers were expressed as a proportion of the relative number detected from the functional ENp
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Fig7: Expression of EN proteins containing mutations in putative phosphorylation sites can induce DNA damage. a Representative western blot analysis of total cell lysates harvested from HeLa cells transiently transfected with the indicated EN putative phosphorylation mutant plasmids. EN is the functional protein and EN- is a non-functional protein containing inactivating mutations (D205A/H230A). Control lanes indicate cells transfected with an empty vector. a Lysates were probed with polyclonal antibodies generated against the human L1 ORF2 endonuclease domain [42, 43], top panel; anti-γH2AX antibodies to detect the phosphorylation of histone H2AX in response to DNA damage, middle panel; and anti-GAPDH to serve as a loading control, bottom panel. b Western blot quantitation. For each sample, the signal detected for ENp was normalized to the signal detected for GAPDH. These relative numbers were expressed as a proportion of the relative number detected from the functional ENp. Asterisk denotes a significant difference in the steady-state levels relative to the functional ENp (t-test, P ≤ 0.05). c Western blot quantitation. For each sample, the signal detected for γH2AX was normalized to the signal detected for GAPDH. These relative numbers were expressed as a proportion of the relative number detected from the functional ENp

Mentions: We have previously reported that expression of the endonuclease domain alone in cultured cells results in a DNA damage response [43]. With the exception of the EN 9m mutant, which was roughly 2-fold higher than the functional ENp, steady-state expression levels were comparable between the functional and mutant proteins (Fig. 7). As previously reported [43], we detected higher steady-state levels of the non-functional ENp (EN-) in comparison to the functional ENp. Similar results were observed with western blot analysis of total protein lysates harvested from 293 cells transiently transfected with the EN expression plasmids (Additional file 9: Figure S6). Western blot analysis detected a γH2AX signal in the total protein lysates from HeLa cells transiently transfected with each of the EN mutant constructs. This result demonstrates that these mutant EN proteins are capable of inducing a DNA damage response (Fig. 7c). Quantitation of the relative γH2AX signals showed that expression of the functional ENp and the EN putative phosphorylation mutant proteins triggered similar levels of H2AX phosphorylation in HeLa cells (i.e., no statistically significant difference was detected) (Fig. 7c).Fig. 7


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 EN proteins containing mutations in putative phosphorylation sites can induce DNA damage. a Representative western blot analysis of total cell lysates harvested from HeLa cells transiently transfected with the indicated EN putative phosphorylation mutant plasmids. EN is the functional protein and EN- is a non-functional protein containing inactivating mutations (D205A/H230A). Control lanes indicate cells transfected with an empty vector. a Lysates were probed with polyclonal antibodies generated against the human L1 ORF2 endonuclease domain [42, 43], top panel; anti-γH2AX antibodies to detect the phosphorylation of histone H2AX in response to DNA damage, middle panel; and anti-GAPDH to serve as a loading control, bottom panel. b Western blot quantitation. For each sample, the signal detected for ENp was normalized to the signal detected for GAPDH. These relative numbers were expressed as a proportion of the relative number detected from the functional ENp. Asterisk denotes a significant difference in the steady-state levels relative to the functional ENp (t-test, P ≤ 0.05). c Western blot quantitation. For each sample, the signal detected for γH2AX was normalized to the signal detected for GAPDH. These relative numbers were expressed as a proportion of the relative number detected from the functional ENp
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig7: Expression of EN proteins containing mutations in putative phosphorylation sites can induce DNA damage. a Representative western blot analysis of total cell lysates harvested from HeLa cells transiently transfected with the indicated EN putative phosphorylation mutant plasmids. EN is the functional protein and EN- is a non-functional protein containing inactivating mutations (D205A/H230A). Control lanes indicate cells transfected with an empty vector. a Lysates were probed with polyclonal antibodies generated against the human L1 ORF2 endonuclease domain [42, 43], top panel; anti-γH2AX antibodies to detect the phosphorylation of histone H2AX in response to DNA damage, middle panel; and anti-GAPDH to serve as a loading control, bottom panel. b Western blot quantitation. For each sample, the signal detected for ENp was normalized to the signal detected for GAPDH. These relative numbers were expressed as a proportion of the relative number detected from the functional ENp. Asterisk denotes a significant difference in the steady-state levels relative to the functional ENp (t-test, P ≤ 0.05). c Western blot quantitation. For each sample, the signal detected for γH2AX was normalized to the signal detected for GAPDH. These relative numbers were expressed as a proportion of the relative number detected from the functional ENp
Mentions: We have previously reported that expression of the endonuclease domain alone in cultured cells results in a DNA damage response [43]. With the exception of the EN 9m mutant, which was roughly 2-fold higher than the functional ENp, steady-state expression levels were comparable between the functional and mutant proteins (Fig. 7). As previously reported [43], we detected higher steady-state levels of the non-functional ENp (EN-) in comparison to the functional ENp. Similar results were observed with western blot analysis of total protein lysates harvested from 293 cells transiently transfected with the EN expression plasmids (Additional file 9: Figure S6). Western blot analysis detected a γH2AX signal in the total protein lysates from HeLa cells transiently transfected with each of the EN mutant constructs. This result demonstrates that these mutant EN proteins are capable of inducing a DNA damage response (Fig. 7c). Quantitation of the relative γH2AX signals showed that expression of the functional ENp and the EN putative phosphorylation mutant proteins triggered similar levels of H2AX phosphorylation in HeLa cells (i.e., no statistically significant difference was detected) (Fig. 7c).Fig. 7

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