Wang Yongming, Wang Jichang, Devaraj Anatharam, Singh Manvendra, Jimenez Orgaz Ana, Chen Jia-Xuan, Selbach Matthias, Ivics Zoltán, Izsvák Zsuzsanna
Max-Delbrück-Center for Molecular Medicine (MDC), Berlin, Germany; State Key Laboratory of Genetic Engineering and Ministry of Education Key Laboratory of Contemporary Anthropology, School of Life Sciences, Fudan University, Shanghai, China.
Max-Delbrück-Center for Molecular Medicine (MDC), Berlin, Germany.
PLoS Genet. 2014 Mar 13;10(3):e1004103. doi: 10.1371/journal.pgen.1004103. eCollection 2014 Mar.
Transposons are discrete segments of DNA that have the distinctive ability to move and replicate within genomes across the tree of life. 'Cut and paste' DNA transposition involves excision from a donor locus and reintegration into a new locus in the genome. We studied molecular events following the excision steps of two eukaryotic DNA transposons, Sleeping Beauty (SB) and piggyBac (PB) that are widely used for genome manipulation in vertebrate species. SB originates from fish and PB from insects; thus, by introducing these transposons to human cells we aimed to monitor the process of establishing a transposon-host relationship in a naïve cellular environment. Similarly to retroviruses, neither SB nor PB is capable of self-avoidance because a significant portion of the excised transposons integrated back into its own genome in a suicidal process called autointegration. Barrier-to-autointegration factor (BANF1), a cellular co-factor of certain retroviruses, inhibited transposon autointegration, and was detected in higher-order protein complexes containing the SB transposase. Increasing size sensitized transposition for autointegration, consistent with elevated vulnerability of larger transposons. Both SB and PB were affected similarly by the size of the transposon in three different assays: excision, autointegration and productive transposition. Prior to reintegration, SB is completely separated from the donor molecule and followed an unbiased autointegration pattern, not associated with local hopping. Self-disruptive autointegration occurred at similar frequency for both transposons, while aberrant, pseudo-transposition events were more frequently observed for PB.
转座子是DNA的离散片段，具有在整个生命之树的基因组中移动和复制的独特能力。“剪切粘贴”DNA转座涉及从供体位点切除并重新整合到基因组中的新位点。我们研究了两种真核DNA转座子——睡美人（SB）和猪尾巴（PB）切除步骤后的分子事件，这两种转座子在脊椎动物物种的基因组操作中被广泛使用。SB起源于鱼类，PB起源于昆虫；因此，通过将这些转座子引入人类细胞，我们旨在监测在一个未接触过的细胞环境中建立转座子-宿主关系的过程。与逆转录病毒类似，SB和PB都不能自我规避，因为很大一部分切除的转座子会在一个称为自整合的自杀过程中重新整合到其自身基因组中。自整合屏障因子（BANF1）是某些逆转录病毒的细胞辅助因子，可抑制转座子自整合，并在含有SB转座酶的高阶蛋白质复合物中被检测到。转座子大小增加会使其自整合的转座敏感性增强，这与较大转座子更高的脆弱性一致。在三种不同的测定中，SB和PB受转座子大小的影响相似：切除、自整合和有效转座。在重新整合之前，SB与供体分子完全分离，并遵循无偏向的自整合模式，与局部跳跃无关。两种转座子的自我破坏自整合发生频率相似，而PB更频繁地观察到异常的假转座事件。
