Abstract
Helitrons are eukaryotic DNA transposons that have profoundly affected genome variability via capture and mobilization of host genomic sequences. Defining their mode of action is therefore important for understanding how genome landscapes evolve. Sequence similarities with certain prokaryotic mobile elements suggest a "rolling circle" mode of transposition, involving only a single transposon strand. Using the reconstituted Helraiser transposon to study Helitron transposition in cells and in vitro, we show that the donor site must be double-stranded and that single-stranded donors will not suffice. Nevertheless, replication and integration assays demonstrate the use of only one of the transposon donor strands. Furthermore, repeated reuse of Helraiser donor sites occurs following DNA synthesis. In cells, circular double-stranded intermediates that serve as transposon donors are generated and replicated by Helraiser transposase. Cell-free experiments demonstrate strand-specific cleavage and strand transfer, supporting observations made in cells.