Fig. 5
From: Helraiser intermediates provide insight into the mechanism of eukaryotic replicative transposition
In vitro transposon-end junction formation and cleavage with purified Helraiser transposase. a Helraiser donor plasmid (pHelR-Cam), used in linear form, and resulting Helraiser circle. Red arrows represent fwd and rev nested primer binding sites; red line: expected size of the PCR product. b PCR detection of Helraiser transposon-end junctions. M marker, Red box marks the position of the expected PCR product. c PCR detection of Helraiser transposon-end junctions ±ATP. d Strand-specific PCR detection of transposon-end junctions generated in vitro by the Helraiser transposase. Lanes 1–3: detection of plus strand. Lanes 4–6: detection of minus strand. e Top: schematic of Helraiser heteroduplex donor plasmid, pHelR(mm)-Cam and resulting Helraiser circle. Red x mismatch position within transposon sequence; red circle position of the mismatch on the donor molecule used in the analysis of the Helraiser circles. Bottom left: PCR detection of the transposon-end junctions generated from the heteroduplex donor. Red arrow indicates PCR product of the expected size spanning the mismatch position. Bottom right: DNA base composition at the mismatch position in the obtained PCR product. Most of the detected junctions arose from the plus strand; four arose from the minus strand. f Top: cleavage of ss 51-mer DNA oligonucleotides representing plus and minus strand of transposon-end junction. Marker (M): 20 bp oligonucleotide corresponding to the expected cleavage product for each strand. Bottom: schematic representation of the ss oligonucleotide used in cleavage reactions. Red arrow indicates the cleavage site. Oligonucleotide sequences are listed in Supplementary table 1
