The autoregulation of a eukaryotic DNA transposon

Abstract

How do DNA transposons live in harmony with their hosts? Bacteria provide the only documented mechanisms for autoregulation, but these are incompatible with eukaryotic cell biology. Here we show that autoregulation of Hsmar1 operates during assembly of the transpososome and arises from the multimeric state of the transposase, mediated by a competition for binding sites. We explore the dynamics of a genomic invasion using a computer model, supported by in vitro and in vivo experiments, and show that amplification accelerates at first but then achieves a constant rate. The rate is proportional to the genome size and inversely proportional to transposase expression and its affinity for the transposon ends. Mariner transposons may therefore resist post-transcriptional silencing. Because regulation is an emergent property of the reaction it is resistant to selfish exploitation. The behavior of distantly related eukaryotic transposons is consistent with the same mechanism, which may therefore be widely applicable.DOI: http://dx.doi.org/10.7554/eLife.00668.001

A single active site in the mariner transposase cleaves DNA strands of opposite polarity

“…A linear DNA fragment encoding the transposon end was titrated with transposase and the complexes were analyzed by gel electrophoresis (Figure 2A). This revealed two single-ended complexes, labeled SEC1 and SEC2, which are similar to the complexes usually observed with other mariner transposases (18,26,28,37). …”

“…The two structural isoforms of SEC2, in which the first (MBP-tagged) or the second (TrxA-tagged) subunit of the dimer is bound in cis to the transposon end, are indicated. The decreasing amount of PEC at the expense of SEC2 with increasing transposase concentration is a predicted feature of our model of mariner autoregulation (28) (see Figure 6A). For a characterization of the single-chain dimer see also Supplementary Figure S1.…”

“…This demonstrates that SEC1 contains a monomer of transposase. We have previously shown that SEC1 arises from dissociation of the PEC during electrophoresis (18,26,28,37). SEC1 is therefore probably not an intermediate of the transposition reaction.…”

“…Transposase concentrations were carefully adjusted to optimal levels because an excess of transposase inhibits the reaction while lower concentrations do not allow complete consumption of the substrate (28). Unless stated otherwise, a 50 μl reaction contained 6.7 nM (1 μg) of the plasmid substrate pRC650 and 20 nM of transposase in 20 mM Tris–HCl buffer pH 8, 100 mM NaCl, 10% glycerol, 2 mM DTT and 2.5 mM MgCl 2 .…”

“…Previous studies have attempted to shed light onto this question by characterizing protein–DNA complexes formed between mariner transposases and the transposon ends (18,26–28). Generally, these have been difficult to interpret and have often led to contradictory conclusions.…”