Transposition without transposase: a spontaneous mutation in bacteria
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Abstract:
Transposition mutations are typically associated with the activities of transposable elements such as transposons and insertion sequences, whose mobility is dependent upon transposase enzymes that catalyze exchanges between element ends and target sites. We describe a single transposition event in which a block of donor sequence is inserted at a target site without the involvement of any known transposase or the ends of any known transposable element. We propose that this is a new type of spontaneous mutation which may be difficult to detect in standard mutant hunts but may be of evolutionary importance.Keywords:
Transposase
Transposition (logic)
Insertion sequence
Tn10
P element
DNA Transposable Elements
Transposase
Tn10
Transposition (logic)
Insertion sequence
P element
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ABSTRACT The movement of the bacterial insertion sequence IS50 and of composite elements containing direct terminal repeats of IS50 involves the two ends of IS50, designated O (outside) and I (inside), which are weakly matched in DNA sequence, and an IS50 encoded protein, transposase, which recognizes the O and I ends and acts preferentially in cis. Previous data had suggested that, initially, transposase interacts preferentially with the O end sequence and then, in a second step, with either an O or an I end. To better understand the cis action of transposase and how IS50 ends are selected, we generated a series of composite transposons which contain direct repeats of IS50 elements. In each transposon, one IS50 element encoded transposase (tnp +), and the other contained a null (tnp -) allele. In each of the five sets of composite transposons studied, the transposon for which the tnp + IS50 element contained its O end was more active than a complementary transposon for which the tnp - IS50 element contained its O end. This pattern of O end use suggests models in which the cis action of transposase and its choice of ends is determined by protein tracking along DNA molecules.
Transposase
Insertion sequence
P element
Tn10
Inverted repeat
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Transposition mutations are typically associated with the activities of transposable elements such as transposons and insertion sequences, whose mobility is dependent upon transposase enzymes that catalyze exchanges between element ends and target sites. We describe a single transposition event in which a block of donor sequence is inserted at a target site without the involvement of any known transposase or the ends of any known transposable element. We propose that this is a new type of spontaneous mutation which may be difficult to detect in standard mutant hunts but may be of evolutionary importance.
Transposase
Transposition (logic)
Insertion sequence
Tn10
P element
DNA Transposable Elements
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ABSTRACT The bacterial transposon Tn5 possesses a regulatory mechanism that allows it to move with higher efficiency when it is first introduced into a cell than after it is established. Tn5 is a composite transposable element containing inverted repeats of two nearly identical elements, IS50R, which encodes the transposase protein necessary for Tn5 movement, and IS50L which contains an ochre mutant allele of the transposase gene. Data presented here show that Tn5 transposition is inhibited about 50-fold in cells of Escherichia coli which already carry IS50R in the multicopy plasmid pBR322. If the cells contain a plasmid carrying either IS50L instead of IS50R, or derivatives of IS50R in which the transposase gene has been mutated, little if any inhibition of Tn5 transposition is found. Although inhibition had previously been hypothesized to require interaction between the products of IS50L and IS50R, our results show that IS50R alone is sufficient to mediate inhibition and suggest that the inhibitor is a product of the transposase gene itself.
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Transposition (logic)
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Insertion sequence
Tn10
Inverted repeat
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Escherichia coli W3110 contains four types of IS1 elements in the chromosome. Using an insertion element entrapping system, we collected 116 IS1 plasmid insertion mutants, which resulted from a minimum of 26 independent IS1 insertion events. All of them had insertions of IS1 of the IS1A (IS1E and IS1G) type. Inspection of the transposase sequences of the four IS1 types and the IS1 of the resistance plasmid R100 showed that two amino acid residues, His-193 and Leu-217 of transposase, might contribute to differential transposability of IS1 elements in W3110. The two amino acid residues of the transposase in IS1A (IS1E and IS1G) were altered separately by site-directed mutagenesis, and each mutant was found to mediate transposition at a frequency about 30-fold lower than that of IS1A (IS1E and IS1G). Thus, the assumption that His-193 and Leu-217 of transposase contribute to differential transposability of IS1 elements in W3110 was confirmed.
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Transposon Tn10 is 9300 bp long and has inverted repeats of insertion sequence IS10 at its ends (Kleckner et al. 1975; Fig. 1). One of these sequences, IS10-Right (IS10-R) encodes a transposase function that acts at the ends of Tn10 to promote transposition of the entire element and acts at the inside and outside ends of IS10-R to promote transposition of the insertion sequence alone (Foster et al. 1981; Halling et al. 1982; see also below). Transposase may also interact with target DNA during insertion when a symmetrical, Tn10-specific target DNA sequence is recognized and staggered cleavages are made to either side (Halling and Kleckner 1982). IS10 transposase is preferentially cis-acting because it does not diffuse freely through the Cell (Morisato et al. 1983).
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Transposition (logic)
Sequence (biology)
DNA Transposable Elements
Inverted repeat
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Tn10
Transposase
P element
Insertion sequence
Insertion
Transposition (logic)
Chloramphenicol acetyltransferase
Transposon mutagenesis
Insertional mutagenesis
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In vitro transposition of ISY100, a bacterial insertion sequence belonging to the Tc1/mariner family
Summary The Synechocystis sp. PCC6803 insertion sequence ISY 100 (IS TcSa ) belongs to the Tc 1 / mariner /IS 630 family of transposable elements. ISY 100 transposase was purified and shown to promote transposition in vitro . Transposase binds specifically to ISY 100 terminal inverted repeat sequences via an N‐terminal DNA‐binding domain containing two helix–turn–helix motifs. Transposase is the only protein required for excision and integration of ISY 100 . Transposase made double‐strand breaks on a supercoiled DNA molecule containing a mini‐ISY 100 transposon, cleaving exactly at the transposon 3′ ends and two nucleotides inside the 5′ ends. Cleavage of short linear substrates containing a single transposon end was less precise. Transposase also catalysed strand transfer, covalently joining the transposon 3′ end to the target DNA. When a donor plasmid carrying a mini‐ISY 100 was incubated with a target plasmid and transposase, the most common products were insertions of one transposon end into the target DNA, but insertions of both ends at a single target site could be recovered after transformation into Escherichia coli . Insertions were almost exclusively into TA dinucleotides, and the target TA was duplicated on insertion. Our results demonstrate that there are no fundamental differences between the transposition mechanisms of IS 630 family elements in bacteria and Tc 1 / mariner elements in higher eukaryotes.
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Inverted repeat
Insertion sequence
Tn10
Transposition (logic)
P element
Bacteriophage Mu
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Transposase
Inverted repeat
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Transposition (logic)
Tn10
P element
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An unusual subset of DNA-binding proteins, termed cis-acting proteins, has been shown to act preferentially at their site of synthesis; the transposases of several bacterial insertion sequences (ISs) fall into this class. The transposase of IS903 exhibits a strong preference for action in cis: complementation of defective transposons in trans occurs at less than 1%. Furthermore, transposition mediated by transposase acting in cis is extremely sensitive to the distance between the 3' end of the transposase gene and the nearest transposon inverted repeat; we find that an insertion of 1 kilobase of DNA reduces transposition to 1-2% of control levels. Here we show that there is a strong correlation between the stability of transposase and its ability to act in trans. We found that the wild-type transposase is a very unstable protein with a physical half-life of about 3 min. However, a transposase-beta-galactosidase fusion protein has a much greater half-life and can act equally well in cis or in trans. In addition, the native transposase is stabilized in lon- strains of Escherichia coli, and, in these protease-deficient strains, trans action of transposase is increased 10- to 100-fold. These results suggest that instability of the IS903 transposase is a major determinant of its cis action and that the La protease, product of the lon gene, is an important determinant of transposase instability.
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Tn10
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Transposition (logic)
Inverted repeat
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