Abstract
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.
Highlights
Mobilization of transposable elements (TEs) is a DNA recombination reaction that can occur either via RNA or DNA intermediates (DNA transposon)
Autointegration events into Zeo or Ori would not be detectable with the assay system, because these regions are required for plasmid propagation and maintenance
This study focuses on molecular events following the excision steps of two eukaryotic DNA transposons, Sleeping Beauty (SB) and PB, derived from fish and insect genomes, respectively
Summary
Mobilization of transposable elements (TEs) is a DNA recombination reaction that can occur either via RNA (retroelement/retrovirus) or DNA intermediates (DNA transposon). In non-replicative, ‘cut and paste’ DNA transposition, the excised transposon relocates from one genomic location to another. The ‘copy and paste’ mobilization of a retroelement/ retrovirus does not include the excision step, but the downstream events of retroviral integration are highly similar to DNA transposition [1] Many DNA transposons are bracketed by terminal inverted repeats (IRs) that contain binding sites for the recombinase, the transposase. The RNA interference (RNAi) machinery in eukaryotes is probably the best-known cellular mechanism that evolved to control transposition [5,6]. Our understanding of the mechanisms and the regulation of transposition in eukaryotes are mostly based on assuming analogies to bacterial transposons [2,7,8]
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