Skip to main content
. 2016 May 25;5:e15537. doi: 10.7554/eLife.15537

Figure 3. Dynamic base flipping of the target adenines.

(a) Target DNA binding in the Mos1 STC, showing the flipped A1 conformation. The unpaired T0 base stacks with the C-1 base of the same strand. See Figure 3—figure supplement 1 for the effect on strand transfer activity of the mutation H122A. (b) Schematic of the TA1 DNA duplex and gel filtration chromatograms of Mos1 transposase (red), TA1 (blue) and the STC (black). UV absorbance at 280 nm (solid line) and 260 nm (dotted line). (c and d) Fluorescence spectroscopy of the 2AP-labelled DNA oligonucleotides TP13 and TP1, shown schematically in (c) and (d) respectively. The A-factor (fractional population) and lifetime of each of the four fluorescence decay components are plotted for TP13 and TP1 alone (black circles and lines) and in the presence of Mos1 transposase (red triangles and lines); and tabulated in Figure 3—source data 1. The steady-state fluorescence emission spectra are inset in each case.

DOI: http://dx.doi.org/10.7554/eLife.15537.010

Figure 3—source data 1. Fluorescence decay parameters for 2AP-containing duplexes, TP13 and TP1, in the absence and presence of Mos1 transposase.
DOI: http://dx.doi.org/10.7554/eLife.15537.011
elife-15537-fig3-data1.docx (31.2KB, docx)
DOI: 10.7554/eLife.15537.011

Figure 3.

Figure 3—figure supplement 1. Strand transfer assay comparing the activity of T216A and H122A/T216A Mos1 transposases.

Figure 3—figure supplement 1.

(a) Denaturing PAGE of the strand transfer reaction products. Lanes 1 and 6 contain markers; lane 2 is without transposase; lane 3 has no target DNA, but integration occurs at the two TA dinucleotides within the IR DNA sequence. (b) Quantification of the 40 nt and 68 nt strand transfer products for each mutant transposase, as a percentage of total DNA.
DOI: http://dx.doi.org/10.7554/eLife.15537.012