Kines Kristine J, Sokolowski Mark, deHaro Dawn L, Christian Claiborne M, Baddoo Melody, Smither Madison E, Belancio Victoria P
Department of Structural and Cellular Biology, Tulane School of Medicine, Tulane Cancer Center and Tulane Center for Aging, New Orleans, LA 70112 USA.
Mob DNA. 2016 Apr 19;7:8. doi: 10.1186/s13100-016-0064-x. eCollection 2016.
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.
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.
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.
人类基因组约17%由长散在核元件1（LINE-1或L1）逆转录转座子组成，它是目前唯一活跃的自主逆转录元件家族。尽管L1元件在数百万年里推动了哺乳动物基因组的进化，但L1的活性也可能具有致突变性并导致人类疾病。L1表达有可能通过逆转录转座和DNA双链断裂（DSB）导致基因组不稳定。此外，L1还导致其他转座元件（如Alu和SVA）诱导的结构基因组变异，这些元件的传播依赖于L1 ORF2蛋白。与L1活性相关的大多数基因组损伤源于ORF2蛋白的核酸内切酶结构域，该结构域切割DNA以准备进行靶标引发的逆转录。
对人类基因组中全长L1基因座的生物信息学分析确定了ORF2核酸内切酶结构域氨基酸序列中的大量突变。其中一些突变存在于预计为细胞激酶磷酸化位点的残基中。我们对ORF2核酸内切酶结构域中的几个假定磷酸化位点进行了突变，并研究了这些突变对全长ORF2蛋白和单独的核酸内切酶结构域（ENp）功能的影响。测试的大多数单点和多点突变对全长ORF2p的表达没有显著影响，也没有改变其驱动Alu逆转录转座的能力。同样，这些突变中的大多数也没有显著改变ENp的表达，或损害其诱导DNA损伤和产生毒性的能力。
总体而言，我们的数据表明，全长ORF2p或单独的ENp在内切酶结构域中可以耐受几个特定的单点和多点突变，而不会分别显著损害它们支持Alu转座或诱导DNA损伤的能力。
