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Query: EC:2.7.10.1 (ERK)
 95,504 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The Escherichia coli insertion sequence, IS2, is a member of the IS3 family of bacterial transposable elements. Its transposase is a fusion protein, OrfAB, made by a programmed -1 translational frameshift near to the end of orfA and just after the start of orfB. We have characterized two major products of IS2 intramolecular transposition, which accumulate in cells that express the IS2 OrfAB fusion protein at elevated levels. The more abundant product is a minicircle composed of the complete IS2 with just a single basepair (occasionally 2bp) separating the two IS ends. In all cases, this basepair is derived from the vector sequence immediately adjacent to the left IS2 end (IRL). The second product is a figure-eight molecule that contains all the IS2 and vector sequences present in the parental plasmid. One DNA strand contains the parental sequences unrearranged. The other contains a single-stranded version of the minicircle junction--the precise 3' end of IRR has been cleaved and joined to a target just outside the 5' end of IRL; the remaining vector sequences have a free 5' end, derived from cleavage at the 3' end of IRR, and a free 3' end, released upon cleavage of the target site adjacent to IRL. We propose that figure-eight molecules are the precursor to IS2 minicircles and that the formation of these two products is the initial step in IS2 intermolecular transposition. This proposed transposition pathway provides a means for a transposase that can cleave only one strand at each IS end to produce simple insertions and avoid forming co-integrates.
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PMID:Two abundant intramolecular transposition products, resulting from reactions initiated at a single end, suggest that IS2 transposes by an unconventional pathway. 930 14

IS3 transposase has been shown to promote production of characteristic circular and linear IS3 molecules from the IS3-carrying plasmid; IS3 circles have the entire IS3 sequence with terminal inverted repeats, IRL and IRR, which are separated by a three base-pair sequence originally flanking either end in the parental plasmid, whereas linear IS3 molecules have three nucleotide overhangs at their 5' ends. Here, we showed that a plasmid carrying an IS3 derivative, which is flanked by different sequences at both ends, generated IS3 circles and linear IS3 molecules owing to the action of transposase. Cloning and sequencing analyses of the linear molecules showed that each had the same 5'-protruding three nucleotide overhanging sequences at both ends, suggesting that the linear molecules were not generated from the parental plasmid by the two double-strand breaks at both end regions of IS3. The plasmid carrying IS3 with a two base-pair mutation in the terminal dinucleotide, which would be required for transposase to cleave the 3' end of IS3, could still generate linear molecules as well as circles. Plasmids bearing an IS3 circle were cleaved by transposase and gave linear molecules with the same 5'-protruding three nucleotide overhanging sequences. These show that the linear molecules are generated from IS3 circles via a double-strand break at the three base-pair intervening sequence. Plasmids carrying an IS3 circle with the two base-pair end mutation still were cleaved by transposase, though with reduced efficiencies, suggesting that IS3 transposase has the ability to cleave not only the 3' end of IS3, but a site three nucleotides from the 5' end of IS3. IS3 circles also were shown to transpose to the target plasmids. The end mutation almost completely inhibited this transposition, showing that the terminal dinucleotides are important for the transfer of the 3' end of IS3 to the target as well as for the end cleavage.
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PMID:Linearization and transposition of circular molecules of insertion sequence IS3. 1055 26

Using a combined in vivo and in vitro approach, we demonstrated that the transposition products generated by IS911 from a dimeric donor plasmid are different from those generated from a plasmid monomer. When carried by a monomeric plasmid donor, free IS911 transposon circles are generated by intra-IS recombination in which one IS end undergoes attack by the other. These represent transposition intermediates that undergo integration using the abutted left (IRL) and right (IRR) ends of the element, the active IRR-IRL junction, to generate simple insertions. In contrast, the two IS911 copies carried by a dimeric donor plasmid not only underwent intra-IS recombination to generate transposon circles but additionally participated in inter-IS recombination. This also creates an active IRR-IRL junction by generating a head-to-tail IS tandem dimer ([IS]2) in which one of the original plasmid backbone copies is eliminated in the formation of the junction. Both transposon circles and IS tandem dimers are generated from an intermediate in which two transposon ends are retained by a single strand joint to generate a figure 8 molecule. Inter-IS figure 8 molecules generated in vitro could be resolved into the [IS]2 form following introduction into a host strain by transformation. Resolution did not require IS911 transposase. The [IS]2 structure was stable in the absence of transposase but was highly unstable in its presence both in vivo and in vitro. Previous studies had demonstrated that the IRR-IRL junction promotes efficient intermolecular integration and intramolecular deletions both in vivo and in vitro. Integration of the [IS]2 derivative would result in a product that resembles a co-integrate structure. It is also shown here that the IRR-IRL junction of the [IS]2 form and derivative structures can specifically target one of the other ends in an intramolecular transposition reaction to generate transposon circles in vitro. These results not only demonstrate that IS911 (and presumably other members of the IS3 family) is capable of generating a range of transposition products, it also provides a mechanistic framework which explains the formation and activity of such structures previously observed for several other unrelated IS elements. This behaviour is probably characteristic of a large number of IS elements.
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PMID:The role of tandem IS dimers in IS911 transposition. 1076 Jan 33

Inverted repeats of insertion sequences (ISs) are indispensable for transposition. We demonstrate that sub-terminal sequences adjacent to the inverted repeats of IS30 are also required for optimal transposition activity. We have developed a cell-free recombination system and showed that the transposase catalyses formation of a figure-of-eight transposition intermediate, where a 2 bp long single strand bridge holds the inverted repeat sequences (IRs) together. This is the first demonstration of the figure-of-eight structure in a non-IS3 family element, suggesting that this mechanism is likely more widely adopted among IS families. We show that the absence of sub-terminal IS30 sequences negatively influences figure-of-eight production both in vivo and in vitro. These regions enhance IR-IR junction formation and IR-targeting events in vivo. Enhancer elements have been identified within 51 bp internal to IRL and 17 bp internal to IRR. In the right end, a decanucleotide, 5'-GAGATAATTG-3', is responsible for wild-type activity, while in the left end, a complex assembly of repetitive elements is required. Functioning of the 10 bp element in the right end is position-dependent and the repetitive elements in the left end act cooperatively and may influence bendability of the end. In vitro kinetic experiments suggest that the sub-terminal enhancers may, at least partly, be transposase-dependent. Such enhancers may reflect a subtle regulatory mechanism for IS30 transposition.
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PMID:Sub-terminal sequences modulating IS30 transposition in vivo and in vitro. 1802 96

Mutants of Clostridium thermocellum that had lost the ability to adhere to microcrystalline cellulose were isolated. Six of them that showed diminished ability to depolymerize crystalline cellulose were selected. Size exclusion chromatography of the proteins from the culture supernatant revealed the loss of the supramolecular enzyme complex, the cellulosome. However, denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis resulted in extracellular protein patterns comparable to those of isolated cellulosomes, except for a missing CipA band. Sequencing of the six mutant cipA genes revealed a new insertion (IS) element, IS1447, belonging to the IS3 family. It was inserted into the cipA reading frame in four different locations: cohesin module 1, two different positions in the carbohydrate binding module, and cohesin module 3. The IS sequences were identical and consisted of a transposase gene and the inverted repeats IRR and IRS. The insertion resulted in an obviously nonspecific duplication of 3 base pairs within the target sequence. This lack of specificity allows transposition without the need of a defined target DNA sequence. Eighteen copies of IS1447 were identified in the genomic sequence of C. thermocellum ATCC 27405. At least one of them can be activated for transposition. Compared to the wild type, the mutant culture supernatant, with a completely defective CipA protein, showed equal specific hydrolytic activity against soluble beta-glucan but a 15-fold reduction in specific activity with crystalline cellulose. These results identify a genetic basis for the synergistic effect of complex formation on crystalline-cellulose degradation.
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PMID:Mutations in the scaffoldin gene, cipA, of Clostridium thermocellum with impaired cellulosome formation and cellulose hydrolysis: insertions of a new transposable element, IS1447, and implications for cellulase synergism on crystalline cellulose. 1840 27

Tight regulation of transposition activity is essential to limit damage transposons may cause by generating potentially lethal DNA rearrangements. Assembly of a bona fide protein-DNA complex, the transpososome, within which transposition is catalysed, is a crucial checkpoint in this regulation. In the case of IS911, a member of the large IS3 bacterial insertion sequence family, the transpososome (synaptic complex A; SCA) is composed of the right and left inverted repeated DNA sequences (IRR and IRL) bridged by the transposase, OrfAB (the IS911-encoded enzyme that catalyses transposition). To characterise further this important protein-DNA complex in vitro, we used different tagged and/or truncated transposase forms and analysed their interaction with IS911 ends using gel electrophoresis. Our results allow us to propose a model in which SCA is assembled with a dimeric form of the transposase. Furthermore, we present atomic force microscopy results showing that the terminal inverted repeat sequences are probably assembled in a parallel configuration within the SCA. These results represent the first step in the structural description of the IS911 transpososome, and are discussed in comparison with the very few other transpososome examples described in the literature.
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PMID:A model for the molecular organisation of the IS911 transpososome. 2055 79