Mizuuchi et al. 10.1073/pnas.0706556104. |
Fig. 8. MuA within transpososomes retains catalytic activity at least 48 h at 30°C. The substrate A depicted in SI Fig. 10 was assembled with MM967 labeled with 32P on the 5' end. The substrate B depicted in SI Fig. 10 was assembled with MM969 labeled with 32P on the 5' end. A total of 100 nM of the labeled substrate was incubated at 30°C with 400 nM MuA in the presence of 5 mM CaCl2 (without Mg2+) in otherwise standard reaction conditions. Under these conditions, transpososome assembly takes place normally, but the chemical reaction does not. At intervals, aliquots were transferred to tubes containing MgCl2 to make 5 mM final concentration and incubated for an additional 2 h to detect the chemical step catalyzed by MuA within the complex. The set of lanes on the left side are reactions with substrate A, and those on the right side are reactions with substrate B, as indicated above the lanes. Samples were analyzed by electrophoresis in a 15% urea-acrylamide gel. The preincubation period (hours) in the calcium buffer is indicated above gel lanes, and the substrate and product bands are marked at the sides. Lanes marked S are unreacted substrates, and DNA length markers are shown in the two outside lanes marked M.
Fig. 9. A Tn10 transposase mutant defective in STC formation efficiently promotes disintegration reactions. The Tn10 disintegration substrate S-53 was assembled into a transpososome resembling the STC using either WT or P167S transposase; the latter is a transposase mutant that is defective in strand transfer but not the earlier steps in transposition. Divalent metal cofactor (5 mM MgCl2 or 0.8 mM MnCl2) was added to the reactions as indicated above the lanes and incubated for 2 or 16 h at 25°C before being applied to an 8% urea-polyacrylamide gel. The 5' terminus of the target OE strand of S-53 contained a 32P label, and the scissile phosphate of the same strand also was labeled with 32P. The internal label permitted detection of a species expected to be the double disintegrant (54 nt) (see figure 1 in ref. 1). Species are labeled as in Fig. 6. DD, double disintegrant.
1. Stewart BJ, Wardle SJ, Haniford DB (2002) EMBO J 21:4380-4390.
Fig. 10. Substrates used in this study. The substrates A-I are depicted with the names of the component oligonucleotides.
SI Text
Oligonucleotides Used.
The oligonucleotides used in this study are summarized in SI Figure 10, and the sequences are listed below. SI Fig S10A depicts substrate A of Fig. 2 and the donor substrate of Fig. 5. SI Fig. 10B shows the components of substrates B-E of Fig. 2. MM969, MM970, or both were omitted for substrates B-D of Fig. 2. The substrate used for the experiments of Fig. 3 was an equal mixture of SI Fig. 10B (unlabeled) and SI Fig. 10C (labeled). SI Fig. 10D was the target DNA for the experiment of Fig. 5. MM1723 was the labeled strand. The phosphorothioate containing strands for the experiment of Fig. 4 were assembled by using the oligonucleotides depicted in SI Fig. 10E. MM1671 with a DMT group on was HPLC purified to separate the Rp and Sp stereoisomers essentially as described (1). After deprotection, 5'-phosphorylation by T4 polynucleotide kinase and ATP, and 3'-end labeling by terminal deoxynucleotidyl transferase and [a-32P]ddATP, the purified strand was ligated to MM1670 by making use of MM1672 as a brace. The ligated strand was purified and annealed with MM965, MM969, and MM970. The phosphorothioate-containing strands for the experiment of Fig. 6 were assembled by using the oligonucleotides depicted in SI Fig. 10F. MM1676 with DMT group on was HPLC-purified to separate the Rp and Sp stereoisomers. After deprotection and 5' phosphorylation by T4 polynucleotide kinase and ATP, the purified strand was ligated to MM1675, which was 5'-end-labeled by T4 polynucleotide kinase and [g-32P]ATP by making use of MM1677 as a brace. The ligated strand was purified and annealed with MM1674. The standard Tn10 disintegration substrate, S-53, used in the experiments of Fig. 6 and SI Fig. 9, is depicted in SI Fig. 10G. The precleaved Tn10 OE and HisG1 Tn10 target DNA used in the experiment of Fig. 7 are depicted in SI Fig. 10 H and I, respectively.
MM965 (71mer), 5'-CCG AGG ATT ACG CCA AGC TGC TGA AGC GGC GCA CGA AAA ACG CGA AAG CGT TTC ACG ATA AAT GCG AAA AC.
MM966 (80mer), 5'-GTT TTC GCA TTT ATC GTG AAA CGC TTT CGC GTT TTT CGT GCG CCG CTT CAT CCG ATC TAG CTC GTG GAC GTA GTC GAG CC.
MM967 (75mer), 5'-GTT TTC GCA TTT ATC GTG AAA CGC TTT CGC GTT TTT CGT GCG CCG CTT CAT CGG AGG AAA ACG TTC TTC GGG GCG.
MM968 (25mer), 5'-GGC TCG ACT ACG TCC ACG AGC TAG A.
MM969 (20mer), 5'-CGC CCC GAA GAA CGT TTT CC.
MM970 (21mer), 5'-p-GCA GCT TGG CGT AAT CCT CGG.
MM1670 (49mer), 5'-GTT TTC GCA TTT ATC GTG AAA CGC TTT CGC GTT TTT CGT GCG CCG CTT C.
MM1671 (26mer), 5'-A-s-T CGG AGG AAA ACG TTC TTC GGG GCG (Trityl ON).
MM1672 (40mer), 5'-GAA GAA CGT TTT CCT CCG ATG AAG CGG CGC ACG AAA AAC G.
MM1673 (75mer), 5'-CGC CCC GAA GAA CGT TTT CCT CCG ATG AAG CGG CGC ACG AAA AAC GCG AAA GCG TTT CAC GAT AAA TGC GAA AAC.
MM1674 (26mer), 5'-p-CTG ATG AAT CCC CTA ATG ATT TTG GT.
MM1675 (25mer), 5'-ACC AAA ATC ATT AGG GGA TTC ATC A.
MM1676 (28mer), 5'-G-s-CG CTG AGT GTG TAA ATT TTA ATT TAC A (Trityl ON).
MM1677 (30mer), 5'-CAC TCA GCG CTG ATG AAT CCC CTA ATG ATT.
MM1723 (61mer), 5'-CGT TCA TTA GCA CAA TCA CAG AAG ACT AGA ATA CAC TCG GAG GAA AAC GTT CTT CGG GGC G.
MM1724 (61mer), 5'-CGC CCC GAA GAA CGT TTT CCT CGG AGT GTA TTC TAG TCT TCT GTG ATT GTG CTA ATG AAC G.
TT53 (53mer), 5'-ACC AAA ATC ATT AGG GGA TTC ATC AGC GCT GAG TGT GTA AAT TTT AAT TTA CA.
NT26 (26mer), 5'-CTG ATG AAT CCC CTA ATG ATT TTG GT.
OE-TS (72mer), 5' -CTG ATG AAT CCC CTA ATG ATT TTA ATA TTA ATC ATA TGC TTA AGC TTG ATA TCC ATC TTC GAA TAT GAT CAG.
OE-BS (72mer), 5' -CTG ATC ATA TTC GAA GAT GGA ATA TCA AGC TTA AGC ATA TGA TTA ATA TTA AAA TCA TTA GGG GAT TCA TCA G.
HisG1-T (50mer), 5'-GCA TAA AAA TTA ATT TAC ACA CTC AGC GCC TGA TTG CGA TGG CGG AAA AC.
HisG1-B (50mer), 5' -TGT TTT CCG CCA TCG CAA TCA GGC GCT GAG TGT GTA AAT TAA TTT TTA TGC.
1. Kennedy AK, Haniford DB, Mizuuchi K (2000) Cell 101:295-305.