(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 …
(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 …
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) …
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 …
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 …
(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 …
Fluorescence decay parameters for 2AP-containing duplexes, TP13 and TP1, in the absence and presence of Mos1 transposase.
(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 …
(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…
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 …
(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 …
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 …
(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 …
(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 …
The Mos1 STC (orange) is superimposed on the post-TS cleavage PEC (PDB ID: 3HOS, lavender).
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), …
Related to Figure 9.
Related to Figure 9.
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 |
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 |