A bend, flip and trap mechanism for transposon integration
- University of Edinburgh, United Kingdom
- EaStCHEM School of Chemistry, United Kingdom
Figures
Figure 1
Mos1 transposition.
(a) Schematic of pathway and complexes formed. Each transposon end has a 28 bp IR sequence (triangle) flanked by the TA target site duplication. First and second strand cleavages (scissors) are …
Figure 2 with 3 supplements
Architecture of the Mos1 strand transfer complex.
(a) Structure of the STC, with transposase subunits (orange and blue), IR DNA (orange and green) and target DNA (magenta and black). Figure 2—figure supplement 1 shows the crystal packing …
Figure 2—figure supplement 1
Stereo views of the difference electron density after molecular replacement.
The Fo-Fc electron density (at 2.3σ) is plotted as a pink mesh with the molecular replacement model: the Mos1 PEC structure (3HOS, chains A to F). (a) Full view of one Mos1 PEC molecule, and (b) …
Figure 2—figure supplement 2
Packing arrangement and DNA interactions in the Mos1 STC crystal lattice.
Four copies of the Mos1 STC are shown. The 5' end of each target DNA strand has a 4 nt overhang, with the self-complementary sequence GGCC, which base pairs with a symmetry related overhang in an …
Figure 2—figure supplement 3
Schematic depiction of the interactions between transposase and DNA in the Mos1 STC structure.
The IR DNA is shown in blue, and the target DNA shown in black (top strand) and magenta (bottom strand). Lines connect the detailed description of the interaction to either a circle (denoting a …
Figure 3 with 1 supplement
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 …
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Figure 3—source data 1
Fluorescence decay parameters for 2AP-containing duplexes, TP13 and TP1, in the absence and presence of Mos1 transposase.
- https://doi.org/10.7554/eLife.15537.011
Figure 3—figure supplement 1
Strand transfer assay comparing the activity of T216A and H122A/T216A Mos1 transposases.
(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 …
Figure 4
Transposase interactions with rotated backbone phosphates stabilise the target DNA.
(a) Target DNA phosphate interactions with catalytic domain residues. The side-chains of R186, W159 and K190 can form hydrogen bonds (dotted lines) with backbone phosphate oxygens of A1 and G2 (dista…
Figure 5
Residues that stabilise the transposition product are required for efficient Mos1 transposition in vitro.
Efficiencies of an in vitro Mos1 hop assay, performed using Mos1 transposase mutants and donor plasmids containing a kanamycin resistance gene flanked by Mos1 inverted repeats, as described …
Figure 6
Plasmid-based transposon cleavage assays.
(a) Schematic of the in vitro plasmid-based Mos1 cleavage assay. (b) Agarose gel showing the products of plasmid-based transposon cleavage assays, for each mutant transposase (Tnp) after 2 hr and …
Figure 7
Alignment of the amino acid sequence of Mos1 with six other mariner transposases and five Tc1-family transposases.
The secondary structure elements of Mos1 transposase in the Mos1 STC are shown above the alignment. A red star below the alignment denotes the position of each of the catalytic acidic residues of …
Figure 8
Base-specific recognition of the flipped adenine.
(a) Close up view of one of the flipped target adenines in the Mos1 STC crystal structure showing the hydrogen bond interactions (dotted cyan lines, distance in Å) with the V214 backbone atoms and …
Figure 9 with 1 supplement
Structural comparison of the Mos1 STC with the pre- and post-TS cleavage Mos1 paired-end complexes.
(a) Orthogonal views of the Mos1 STC (orange) superimposed on the pre-TS cleavage PEC (PDB ID: 4U7B, green): r.m.s.d. over all transposase backbone atoms, 1.2 Å. Video 1 and video 2 show the …
Figure 9—figure supplement 1
Structural comparison of the Mos1 STC with the post-TS cleavage Mos1 paired-end complex.
The Mos1 STC (orange) is superimposed on the post-TS cleavage PEC (PDB ID: 3HOS, lavender).
Figure 10
A proposed mechanism for Mos1 transposon integration incorporates target DNA bending and trapping of flipped target adenines.
Schematic representation of key features of the STC (right) and the proposed target capture complex (left), with transposase subunits (orange and blue). Filled circles represent residues W159 (W), …
Videos
Video 1
Morphing of the Mos1 transposase conformation in the pre-TS cleavage PEC (PDB ID: 4U7B) into the Mos1 STC conformation.
Related to Figure 9.
Video 2
Morphing of the Mos1 transposase conformation in the post-cleavage PEC (PDB ID: 3HOS) into the Mos1 STC conformation.
Related to Figure 9.
Tables
Table 1
Sequences of oligonucleotides used in the crystallisation, target integration and fluorescence experiments. The target TA dinucleotide (and its variants) are highlighted in bold. The adenine …
| Name | Sequence | Length (nt) |
|---|---|---|
| Crystallisation of STC | ||
| TS | 5' AAA CGA CAT TTC ATA CTT GTA CAC CTG ATA GCA GTG | 36 |
| NTS | 5' GGT GTA CAA GTA TGA AAT GTC GTT T | 25 |
| target DNA | 5' GGC CCA CTG C | 10 |
| Target Integration Assays | ||
| TS IR DNA | 5' AAA CGA CAT TTC ATA CTT GTA CAC CTG A | 28 |
| TS 5' labelled IR DNA | 5' IR700 / AAA CGA CAT TTC ATA CTT GTA CAC CTG A | 28 |
| NTS IR DNA | 5' GGT GTA CAA GTA TGA AAT GTC GTT T | 25 |
| TA target DNA (top strand) | 5' AGC AGT GCA CTA GTG CAC GAC CGT TCA AAG CTT CGG AAC GGG ACA CTG TT | 50 |
| TA target DNA (bottom strand) | 5' AAC AGT GTC CCG TTC CGA AGC TTT GAA CGG TCG TGC ACT AGT GCA CTG CT | 50 |
| TP target DNA (top strand) | 5' AGC AGT GCA CTP GTG CAC GAC CGT TCA AAG CTT CGG AAC GGG ACA CTG TT | 50 |
| TP target DNA (bottom strand) | 5' AAC AGT GTC CCG TTC CGA AGC TTT GAA CGG TCG TGC ACT PGT GCA CTG CT | 50 |
| TD target DNA (top strand) | 5' AGC AGT GCA CTD GTG CAC GAC CGT TCA AAG CTT CGG AAC GGG ACA CTG TT | 50 |
| TD target DNA (bottom strand) | 5' AAC AGT GTC CCG TTC CGA AGC TTT GAA CGG TCG TGC ACT DGT GCA CTG CT | 50 |
| SD target DNA (top strand) | 5' AGC AGT GCA CSD GTG CAC GAC CGT TCA AAG CTT CGG AAC GGG ACA CTG TT | 50 |
| SD target DNA (bottom strand) | 5' AAC AGT GTC CCG TTC CGA AGC TTT GAA CGG TCG TGC ACS DGT GCA CTG CT | 50 |
| Fluorescence experiments | ||
| TS_P1 | 5' AAA CGA CAT TTC ATA CTT GTA CAC CTG AtP gca gtg gac gta ggc c | 46 |
| TS_P13 | 5' AAA CGA CAT TTC ATA CTT GTA CAC CTG Ata gca gtg gac gtP ggc c | 46 |
| TS_A1 | 5' AAA CGA CAT TTC ATA CTT GTA CAC CTG Ata gca gtg gac gta ggc c | 46 |
| NTS | 5' GGT GTA CAA GTA TGA AAT GTC GTT T | 25 |
| Target_16 | 5' g gcc tac gtc cac tgc | 16 |
Table 2
X-ray diffraction and refinement statistics.
| Crystal | Mos1 Strand transfer complex | |
|---|---|---|
| PDB ID | 5HOO | |
| Space group | C121 | |
| Cell dimensions | a = 256.3 Å b = 58.9 Å c = 110.2 Å α = 90.0°, β = 94.9°, γ = 90.0° | |
| Wavelength (Å) | 0.9795 | |
| Average mosaicity | 0.22 | |
| Overall | Outer shell | |
| Resolution (Å) | 86.99–3.29 | 3.52–3.29 |
| Rsymm | 0.077 | 0.152 |
| Total observations | 78358 | 14630 |
| Unique observations | 25201 | 4479 |
| < I>/σ<I> | 8.1 | 3.3 |
| Correlation CC | 0.927 | 0.996 |
| Completeness (%) | 99.6 | 99.5 |
| Multiplicity | 3.1 | 3.3 |
| Rwork | 0.243 | |
| Rfree (5.21% of reflections) | 0.279 | |
| R.m.s. deviations: Bond Length (Å) Bond Angle (deg) Chiral volume (Å) | 0.0077 1.2072 0.0785 | |
| Average B factor (Å2) | 74.0 | |
| Ramachandran plot: Core (%) Allowed (%) Outliers (%) | 90.8 9.2 0 | |
