Suicidal Autointegration of <i>Sleeping Beauty</i> and <i>piggyBac</i> Transposons in Eukaryotic Cells

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（Transposons）是一类离散的DNA片段，其显著特征是能够在整个生命之树的各类生物的基因组中移动并复制。“剪切-粘贴”型DNA转座（DNA transposition）指从供体位点切除转座子，并将其重新整合到基因组的新位点中。本研究针对两种广泛应用于脊椎动物基因组操作的真核生物DNA转座子——睡美人（Sleeping Beauty, SB）和piggyBac（PB）——的切除后分子事件展开了研究。SB源自鱼类，PB源自昆虫；因此，我们通过将这两种转座子引入人类细胞，旨在监控在未接触过此类转座子的初始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均无法实现自我规避：大量切除后的转座子会重新整合回其自身的基因组中，这一自杀式过程被称为自身整合（autointegration）。自身整合屏障因子（Barrier-to-autointegration factor, BANF1）作为部分逆转录病毒的细胞辅助因子，能够抑制转座子的自身整合，且该因子可在含有SB转座酶的高级蛋白质复合物中被检测到。转座子的长度增加会提升其发生自身整合的易感性，这与更大尺寸的转座子具有更高的易损性相一致。在切除反应、自身整合反应以及功能性转座反应这三种不同的检测实验中，SB和PB均受到转座子尺寸的相似影响。在重新整合之前，SB会完全脱离供体DNA分子，并呈现出无偏向性的自身整合模式，未发生局部跳跃现象。两种转座子的自我破坏性自身整合发生频率相近，而PB则更频繁地出现异常的伪转座事件。