Progressive Structural Transitions within Mu Transpositional Complexes Katsuhiko Yanagihara, Kiyoshi Mizuuchi  Molecular Cell  Volume 11, Issue 1, Pages 215-224 (January 2003) DOI: 10.1016/S1097-2765(02)00796-7

Figure 1 Mu Transpososomes and Structural Organization of MuA Variants (A) Mu transpososomes contain a pair of Mu end DNA fragments and a tetramer of MuA. The two successive chemical steps, donor cleavage and strand transfer, convert the stable synaptic complex (SSC) to the cleaved donor complex (CDC), and then to the strand transfer complex (STC). (B) MuA comprises three structural domains, each of which consists of subdomains. Two of the MuA-GFP fusion proteins, (77-615)-EGFP and EGFP-(77-615), are also depicted. Molecular Cell 2003 11, 215-224DOI: (10.1016/S1097-2765(02)00796-7)

Figure 2 FRET Signal Associated with Transpososome Assembly (A) Gel-shift assay. Precleaved R1-R2 DNA substrate was used. The presence/absence of Mg2+ and DMSO is indicated at the top. The electrophoresis was done in the presence (lower panel) or in the absence (upper panel) of heparin. Band assignments are shown on the right. (B) MuA tetramer cross-linking. The presence/absence of DNA, Mg2+, and DMSO is shown at the top. Cross-linking was done in the presence (lower panel) or absence (upper panel) of heparin. Samples were analyzed by SDS-PAGE, and proteins were visualized by Coomassie G-250. Band assignments are shown on the right. (C) Emission spectrum of reaction mixtures containing equimolar MuA77-615-ECFP and MuA77-615-EYFP. Complete reaction (red squares) followed our standard conditions. Other reactions were in the absence of Mg2+ and DMSO (blue triangles) or DNA (green circles). FRET is recognized as a decrease of emission from ECFP around 475 nm and an increase of emission from EYFP around 525 nm. (D) Emission spectrum of reaction mixtures containing equimolar MuA77-615-ECFP and MuA77-615-EYFP. Reactions were performed with regular precleaved DNA (red squares), precleaved DNA with mutated R2 sequence (open diamonds), precleaved DNA with mutated R1 sequence (crosses), precleaved DNA with mutated R1 and R2 sequence (black triangles), or without DNA (green circles). (E) Emission spectrum of reaction mixtures containing equimolar MuA77-605-ECFP and MuA77-605-EYFP. Reactions were performed with (red squares) or without (green circles) precleaved substrate DNA. (F) Same as (E), except MuA77-575-ECFP and MuA77-575-EYFP were used. Molecular Cell 2003 11, 215-224DOI: (10.1016/S1097-2765(02)00796-7)

Figure 3 Unstable Transpososomes Are Formed in the Absence of the Recombination Substrate Site (A) Transpososome assembly with Mu end DNA substrates with 3′ and/or 5′ recessed ends was assessed by FRET, protein cross-linking in the presence of heparin, and gel-shift assays. The substrates are depicted above, starting with the standard precleaved Mu end DNA 1. The transferred strand with the 3′ end pointing up is drawn on the left side and the nontransferred strand on the right in each column. The MuA binding sites R1 and R2, and the last retained nucleotide positions are indicated. Detection of transpososome assembly by each method was as follows. FRET experiment: +, >7% FRET; −, <2% FRET. Cross-linking experiment: +, ≥50% of the MuA cross-linked to tetramer or higher multimers; −, <10% cross-linking. Gel-shift experiment: +, >20% substrate DNA conversion to heparin-resistant complex; −, <3% conversion. (B) The effect of DMSO concentration on the stability of preformed transpososomes. FRET experiments were performed using the Mu end DNA 1 or Mu end DNA 9 depicted in (A). After the reaction, the samples were split into three aliquots. Two aliquots were diluted 3-fold with diluent that did or did not include DMSO, and one was saved as a nondilution control. The emission of the diluted samples was corrected by a three times longer integration time. The reaction without DMSO was used as the control for no transpososome assembly. Molecular Cell 2003 11, 215-224DOI: (10.1016/S1097-2765(02)00796-7)

Figure 4 The Effects of the Nontransferred Strand Length on the Activities of Transpososome (A) The cleavage activity of transpososomes with DNA having different lengths of nontransferred strand. The DNAs used are depicted with the top strand as the nontransferred strand. Mu end DNA fragments had 5 nt flanking DNA on the transferred strand and the nontransferred strand truncated at the 5′ end. The DNA was labeled with fluorescein at the end of the flanking segment on the transferred strand. After reaction, DNA was purified, and a portion of samples was analyzed by 12% urea-polyacrylamide gel electrophoresis, followed by scanning of the gel with a Molecular Dynamics 8600 with a 526 SP filter. Uncleaved substrate was 56 nt, while the cleaved product was 5 nt. (B) The strand transfer activity of transpososomes formed with substrate DNA having different lengths of nontransferred strand. The DNAs used are depicted on the top. Mu end DNA fragments had precleaved 3′ ends on the transferred strand and a truncation at the 5′ end of the nontransferred strand. After transpososome assembly, φX174 RFI DNA was added to the assembly reaction mixture as target DNA. Two-ended strand transfer products migrated at the position of liner φX174, and one-ended strand transfer products comigrated with nicked φX174 circles. Molecular Cell 2003 11, 215-224DOI: (10.1016/S1097-2765(02)00796-7)

Figure 5 DNA Deformation around the Mu-Flanking DNA Junction Upon Transpososome Assembly (A) Position of 2-aminopurine in the substrate DNA. This DNA has two MuA binding sites, R1 and R2, and 9 bp AT flanking DNA. The nucleotides that were replaced by 2-aminopurine are shown in red-shaded boxes, and the positions are numbered. (B) The fluorescence of 2-aminopurine, incorporated at different positions in the substrate DNA, under different reaction conditions. Positions of 2-aminopurine are indicated on the top of each panel. Reaction conditions are shown below. Control measurements were in the absence of MuA, metals, and/or DMSO. Inclusion of DMSO or metals in the absence of MuA affected fluorescence emission by less than 10%. Molecular Cell 2003 11, 215-224DOI: (10.1016/S1097-2765(02)00796-7)