The importance of L1 ORF2p cryptic sequence to ORF2p fragment-mediated cytotoxicity

Christian, Claiborne M.; Kines, Kristine J.; Belancio, Victoria P.

doi:10.1080/2159256x.2016.1198300

Cited by 4 publications

(5 citation statements)

References 26 publications

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“…This response to EN expression is notably dampened when an ORF2p fragment containing the EN, Cry, and Z sequences is expressed, even though the ENCryZ protein accumulates to higher steady-state levels than the EN protein by 19 h post-induction (Figure 5). This observation is consistent with our previously published data [32,69] that the Cryptic sequence within the human ORF2p may suppress L1 endonuclease function via a yet unknown mechanism. The lack of detectable increase in the γH2AX signal in cells expressing L1 or ORF2p EN+RT-(Figures 2 and 3) could be explained by either rapid repair of the EN-induced nicks in the absence of the functional RT or by the aforementioned suppression of EN activity in the context of the full-length ORF2 protein lacking a functional RT as previously suggested [69].…”

Section: Discussionsupporting

confidence: 93%

“…This observation is consistent with our previously published data [32,69] that the Cryptic sequence within the human ORF2p may suppress L1 endonuclease function via a yet unknown mechanism. The lack of detectable increase in the γH2AX signal in cells expressing L1 or ORF2p EN+RT-(Figures 2 and 3) could be explained by either rapid repair of the EN-induced nicks in the absence of the functional RT or by the aforementioned suppression of EN activity in the context of the full-length ORF2 protein lacking a functional RT as previously suggested [69]. It is worth noting that the two scenarios are not mutually exclusive.…”

Section: Discussionsupporting

confidence: 93%

“…The involvement of the EN domain in causing DNA breaks and eliciting cellular DNA damage response (DDR) has been reported using the detection of phosphorylated H2AX, COMET, or toxicity assays [32,65,69,70]. The histone H2AX is a fairly common component of the chromatin of mammalian cells, with about 1 in 40 to 1 in 4 histones being H2AX, depending on the cell type [71].…”

Section: Introductionmentioning

confidence: 99%

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Large Deletions, Cleavage of the Telomeric Repeat Sequence, and Reverse Transcriptase-Mediated DNA Damage Response Associated with Long Interspersed Element-1 ORF2p Enzymatic Activities

Kines,

Sokolowski,

DeFreece

et al. 2024

Genes

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L1 elements can cause DNA damage and genomic variation via retrotransposition and the generation of endonuclease-dependent DNA breaks. These processes require L1 ORF2p protein that contains an endonuclease domain, which cuts genomic DNA, and a reverse transcriptase domain, which synthesizes cDNA. The complete impact of L1 enzymatic activities on genome stability and cellular function remains understudied, and the spectrum of L1-induced mutations, other than L1 insertions, is mostly unknown. Using an inducible system, we demonstrate that an ORF2p containing functional reverse transcriptase is sufficient to elicit DNA damage response even in the absence of the functional endonuclease. Using a TK/Neo reporter system that captures misrepaired DNA breaks, we demonstrate that L1 expression results in large genomic deletions that lack any signatures of L1 involvement. Using an in vitro cleavage assay, we demonstrate that L1 endonuclease efficiently cuts telomeric repeat sequences. These findings support that L1 could be an unrecognized source of disease-promoting genomic deletions, telomere dysfunction, and an underappreciated source of chronic RT-mediated DNA damage response in mammalian cells. Our findings expand the spectrum of biological processes that can be triggered by functional and nonfunctional L1s, which have impactful evolutionary- and health-relevant consequences.

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Section: Discussionsupporting

confidence: 93%

Section: Discussionsupporting

confidence: 93%

Section: Introductionmentioning

confidence: 99%

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Large Deletions, Cleavage of the Telomeric Repeat Sequence, and Reverse Transcriptase-Mediated DNA Damage Response Associated with Long Interspersed Element-1 ORF2p Enzymatic Activities

Kines,

Sokolowski,

DeFreece

et al. 2024

Genes

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“…This region contains a conserved WD pair that is vital to the complementary DNA (cDNA) synthesis step of TPRT (Christian et al 2016a). The Cry region may be involved in modulating the EN function (Christian et al 2016b). It also contains a putative proliferating cell nuclear antigen (PCNA)-binding domain important for retrotransposition (Christian et al 2016a).…”

mentioning

confidence: 99%

Involvement of Conserved Amino Acids in the C-Terminal Region of LINE-1 ORF2p in Retrotransposition

et al. 2017

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Long interspersed element 1 (L1) is the only currently active autonomous retroelement in the human genome. Along with the parasitic SVA and short interspersed element Alu, L1 is the source of DNA damage induced by retrotransposition: a copy-and-paste process that has the potential to disrupt gene function and cause human disease. The retrotransposition process is dependent upon the ORF2 protein (ORF2p). However, it is unknown whether most of the protein is important for retrotransposition. In particular, other than the Cys motif, the C terminus of the protein has not been intensely examined in the context of retrotransposition. Using evolutionary analysis and the Alu retrotransposition assay, we sought to identify additional amino acids in the C terminus important for retrotransposition. Here, we demonstrate that Gal4-tagged and untagged C-terminally truncated ORF2p fragments possess residual potential to drive Alu retrotransposition. Using sight-directed mutagenesis we identify that while the Y1180 amino acid is important for ORF2p- and L1-driven Alu retrotransposition, a mutation at this position improves L1 retrotransposition. Even though the mechanism of the contribution of Y1180 to Alu and L1 mobilization remains unknown, experimental evidence rules out its direct involvement in the ability of the ORF2p reverse transcriptase to generate complementary DNA. Additionally, our data support that ORF2p amino acids 1180 and 1250-1262 may be involved in the reported ORF1p-mediated increase in ORF2p-driven Alu retrotransposition.

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“…However, it is possible that it contributes to some of the symptoms or to the progression of these diseases. In addition to the potentially deleterious effects that might be caused by somatic retrotransposon insertions (particularly those that disrupt genes), the endonuclease of L1 ORF2p has the potential to cause DNA damage even in the absence of a successful retrotransposition event (Kines et al, 2014;Christian et al, 2016). Likewise, DNA-mediated damage at integration sites appears to be targeted by ATM, and additional DNA damage/repair pathways also may send somatic cells towards apoptosis, necrosis, or senescence.…”

Section: Ataxia-telangiectasia (At)mentioning

confidence: 99%

Aberrantly High Levels of Somatic LINE-1 Expression and Retrotransposition in Human Neurological Disorders

Terry

Devine

2020

Front. Genet.

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Retrotransposable elements (RTEs) have actively multiplied over the past 80 million years of primate evolution, and as a consequence, such elements collectively occupy ~ 40% of the human genome. As RTE activity can have detrimental effects on the human genome and transcriptome, silencing mechanisms have evolved to restrict retrotransposition. The brain is the only known somatic tissue where RTEs are de-repressed throughout the life of a healthy human and each neuron in specific brain regions accumulates up to ~13.7 new somatic L1 insertions (and perhaps more). However, even higher levels of somatic RTE expression and retrotransposition have been found in a number of human neurological disorders. This review is focused on how RTE expression and retrotransposition in neuronal tissues might contribute to the initiation and progression of these disorders. These disorders are discussed in three broad and sometimes overlapping categories: 1) disorders such as Rett syndrome, Aicardi-Goutières syndrome, and ataxia-telangiectasia, where expression/retrotransposition is increased due to mutations in genes that play a role in regulating RTEs in healthy cells, 2) disorders such as autism spectrum disorder, schizophrenia, and substance abuse disorders, which are thought to be caused by a combination of genetic and environmental stress factors, and 3) disorders associated with age, such as frontotemporal lobar degeneration (FTLD), amyotrophic lateral sclerosis (ALS), and normal aging, where there is a time-dependent accumulation of neurological degeneration, RTE copy number, and phenotypes. Research has revealed increased levels of RTE activity in many neurological disorders, but in most cases, a clear causal link between RTE activity and these disorders has not been well established. At the same time, even if increased RTE activity is a passenger and not a driver of disease, a detrimental effect is more likely than a beneficial one. Thus, a better understanding of the role of RTEs in neuronal tissues likely is an important part of understanding, preventing, and treating these disorders.

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The importance of L1 ORF2p cryptic sequence to ORF2p fragment-mediated cytotoxicity

Cited by 4 publications

References 26 publications

Large Deletions, Cleavage of the Telomeric Repeat Sequence, and Reverse Transcriptase-Mediated DNA Damage Response Associated with Long Interspersed Element-1 ORF2p Enzymatic Activities

Large Deletions, Cleavage of the Telomeric Repeat Sequence, and Reverse Transcriptase-Mediated DNA Damage Response Associated with Long Interspersed Element-1 ORF2p Enzymatic Activities

Involvement of Conserved Amino Acids in the C-Terminal Region of LINE-1 ORF2p in Retrotransposition

Aberrantly High Levels of Somatic LINE-1 Expression and Retrotransposition in Human Neurological Disorders

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