Characteristics of MuA transposase-catalyzed processing of model transposon end DNA hairpin substrates

Abstract

Bacteriophage Mu uses non-replicative transposition for integration into the host's chromosome and replicative transposition for phage propagation. Biochemical and structural comparisons together with evolutionary considerations suggest that the Mu transposition machinery might share functional similarities with machineries of the systems that are known to employ a hairpin intermediate during the catalytic steps of transposition. Model transposon end DNA hairpin substrates were used in a minimal-component in vitro system to study their proficiency to promote Mu transpososome assembly and subsequent MuA-catalyzed chemical reactions leading to the strand transfer product. MuA indeed was able to assemble hairpin substrates into a catalytically competent transpososome, open the hairpin ends and accurately join the opened ends to the target DNA. The hairpin opening and transposon end cleavage reactions had identical metal ion preferences, indicating similar conformations within the catalytic center for these reactions. Hairpin length influenced transpososome assembly as well as catalysis: longer loops were more efficient in these respects. In general, MuA's proficiency to utilize different types of hairpin substrates indicates a certain degree of flexibility within the transposition machinery core. Overall, the results suggest that non-replicative and replicative transposition systems may structurally and evolutionarily be more closely linked than anticipated previously.

Figure 1

Donor DNA substrates. (A) Substrate schematic representation. All the Mu-specific substrates contain ∼50 bp, starting from the Mu R-end and including the MuA-binding sites R1 and R2 (22). These substrates also include a flanking DNA region, which can be in duplex DNA, linear single strand or in a hairpin loop configuration. Some substrates contain nucleotide substitutions close to the transposon end or within the loop region [see (B)]. The depicted 16 bp flanking sequence has been shown earlier to support efficient catalysis by MuA in an in vitro model substrate assay (22), and the standard loop sequences are derived from that same sequence by joining the endmost Mu-specific 3′-adenine to various nucleotide positions in the opposite strand of the duplex. The Mu end cleavage point is indicated by a solid arrow and the position of the radioactive label by an asterisk. The open arrow shows the position of the nick present in most substrates [see (B)]. (B) Substrate characteristics. The donor substrates were made by annealing one or two oligonucleotides to form double-stranded species as indicated. Most of the hairpin substrates were generated from two oligonucleotides, and these substrates therefore contain a nick [within the R1 MuA-binding site, see (A)]. This nick does not interfere with in vitro transposition reactions as indicated by a direct comparison of reactions with the nicked and corresponding unnicked substrate (compare lanes 4 and 12 in Figure 5). The endmost nucleotides of the transposon DNA are shown in bold, and flanking nucleotides are indicated in regular font. The rHP4 substrate includes a randomly chosen non-Mu sequence with two conserved base pairs mimicking the transposon end.

Figure 2

MuA transposase catalyzes the processing of model DNA hairpin substrates. (A) Reaction schematic representation. As with standard uncleaved and precleaved model substrates (22), model hairpin substrates and MuA protein assemble the tetrameric Mu transpososome that catalyzes the subsequent reactions. Upon addition of MgCl₂, MuA first catalyzes a hydrolytic strand cleavage reaction (hairpin opening) and then executes a strand transfer reaction, in which the target becomes joined to transposon DNA. The end products include circular or linear target molecules, depending on whether one or two transposon ends become joined to the target, respectively. The radiolabel included in the donor DNA fragment will be incorporated in the DNA product, providing an easy assay for catalysis (–27). (B) Coupled cleavage and strand transfer reaction. HP4 substrate (and PC4 for control) was incubated in the presence of MuA (upper panel), or MuA and MuB (lower panel) for 1–6 h. The strand transfer reaction products (SEPs and DEPs) were analyzed by agarose gel electrophoresis and autoradiography. The unreacted donor substrates that migrate close to the bottom of the gel are not shown. To increase the yield of reaction products, 3-fold scaled up (3×) reactions were used with the HP4 donor, whereas PC4 reactions were 1-fold (1×) for convenient identification of reaction products (Materials and Methods).

Figure 3

Utilization of various metal ions for the catalysis of hairpin processing. In vitro transposition reactions were performed with the indicated substrates by first assembling complexes in the absence of metal ions and then initiating reaction chemistry by adding MgCl₂, MnCl₂ or CaCl₂ (Materials and Methods). The reaction products, SEPs and DEPs, were analyzed by agarose gel electrophoresis and autoradiography following a 3 h incubation in the presence of metal ion.

Figure 4

Reactions of mixed transpososomes. (A) Schematic representation of mixed complex analysis. When unlabeled precleaved substrate (PC4) and 5′-labeled hairpin substrate (HP4) are mixed, four types of transpososomes will be assembled to generate strand transfer products. Only those reaction products containing the label originating from the use of the HP4 donor will be detectable by autoradiography. (B) Analysis of reaction products of mixed HP4 and PC4 donor complexes. Labeled HP4 and unlabeled PC4 donor substrates were used in the indicated ratios to assemble mixed complexes in standard 1-fold in vitro transposition reactions. Transpososomes were first assembled without divalent metal ions for 1 h, after which MgCl₂ was added (Materials and Methods), and samples were withdrawn after incubation for 0, 1 and 6 h. Strand transfer products (DEPs and SEPs) were analyzed by agarose gel electrophoresis and autoradiography.

Figure 5

Effect of hairpin loop length and sequence on stable complex formation and catalysis. (A) Complex formation. Complexes with hairpin (3-fold scaled up reactions) and precleaved (1-fold reactions) substrates were assembled for 1 h in the absence of metal ions, and the reaction products were analyzed by native agarose gel electrophoresis and autoradiography. Primarily, the assay detects transpososomes but may also detect assembly and/or disassembly intermediates. The slower migrating complexes (indicated by C1) include transpososomes. (B) Analysis of strand transfer products. Target DNA (pUC19) and MgCl₂ were added to the assembly reactions to allow catalysis (Materials and Methods), and the reactions were incubated for 6 h prior to analysis of strand transfer products (as in Figure 4). (C) Tabulation of data from (A) and (B). Only a minor portion of the substrates were converted to reaction products (in most cases <1%). The levels of the detected products are based on visual inspection and indicated by a scale of four categories: (−), no products; (+), low level; (++), medium level; (+++), high level. Note that the HP4 substrate did not produce appreciable amounts of DEPs in this experiment, whereas in a similar experiment seen in Figure 2B it did. This apparent discrepancy is due to lower level of radioactivity in the substrate.

Figure 6

Analysis of hairpin opening products. (A) Effect of hairpin loop length. Transposition reactions were performed as in Figure 5. Hairpin opening products of hairpin donors having variable loop length were analyzed by denaturing urea–PAGE and visualized by autoradiography. The length of the expected opening product is 50 nt. The secondary bands seen in several lanes probably represent DNA synthesis impurities or they (mutually not exclusively) may represent DNA migrating in several major conformations, possibly influenced by the presence of hairpin ends. Minor products seen below 50 nt products (lanes 6 and 7) most probably represent cleavage from the (n − 1) synthesis impurities. Several bands seen in the top part of lane 1 represent strand transfer products. (B) Kinetics of hairpin opening reaction. Transposition reaction with HP13A donor was performed, and samples (taken after incubation of 0, 10, 30 and 90 min) were analyzed by urea–PAGE and autoradiography.