Role of the IS50 R proteins in the promotion and control of Tn5 transposition

IS50R, the inverted repeat sequence of Tn5 which is responsible for supplying functions that promote and control Tn5 transposition, encodes two polypeptides that differ at their N terminus. Frameshift, in-frame deletion, nonsense, and missense mutations within the N terminus of protein 1 (which is not present in protein 2) were isolated and characterized. The properties of these mutations demonstrate that protein 1 is absolutely required for Tn5 transposition. None of these mutations affected the inhibitory activity of IS50, confirming that protein 2 is sufficient to mediate inhibition of Tn5 transposition. The effects on transposition of increasing the amount of protein 2 (the inhibitor) relative to protein 1 (the transposase) were also analyzed. Relatively large amounts of protein 2 were required to see a significant decrease in the transposition frequency of an element. In addition, varying the co-ordinate synthesis of the IS50 R proteins over a 30-fold range had little effect on the transposition frequency. These studies suggest that neither the wild-type synthesis rate of protein 2 relative to protein 1 nor the amount of synthesis of both IS50 R proteins is the only factor responsible for controlling the transposition frequency of a wild-type Tn5 element in Escherichia coli.     

Functional domains of the IS1 transposase: analysis in vivo and in vitro

The IS1 bacterial insertion sequence family, considered to be restricted to Enterobacteria, has now been extended to other Eubacteria and to Archaebacteria, reviving interest in its study. To analyse the functional domains of the InsAB' transposase of IS1A, a representative of this family, we used an in vivo system which measures IS1-promoted rescue of a temperature-sensitive pSC101 plasmid by fusion with a pBR322::IS1 derivative. We also describe the partial purification of the IS1 transposase and the development of several in vitro assays for transposase activity. These included a DNA band shift assay, a transposase-mediated cleavage assay and an integration assay. Alignments of IS family members (http://www-is.biotoul.fr) not only confirmed the presence of an N-terminal helix-turn-helix and a C-terminal DDE motif in InsAB', but also revealed a putative N-terminal zinc finger. We have combined the in vitro and in vivo tests to carry out a functional analysis of InsAB' using a series of site-directed InsAB' mutants based on these alignments. The results demonstrate that appropriate mutations in the zinc finger and helix-turn-helix motifs result in loss of binding activity to the ends of IS1 whereas mutations in the DDE domain are affected in subsequent transposition steps but not in end binding.     

Linearization and transposition of circular molecules of insertion sequence IS3.

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.     

Linearity versus Nonlinearity of Offspring-Parent Regression: An Experimental Study of Drosophila Melanogaster

IS10 transposase mediates excision and integration reactions in Tn10/IS10 transposition. Mutations in IS10 transposase that specifically block integration have previously been identified; however, the mechanism by which these mutations block integration has not been established. One approach to defining the basis of this block is to identify ways in which the original defect can be corrected. The approach we have taken toward this end has been to isolate and characterize intragenic second site suppressors to two different integration-defective mutants. Of the second site suppressors identified, one, CY134, is of particular interest for two reasons. First, it suppresses at least seven different mutations that confer an integration-defective phenotype. Interestingly, these mutations map in two separate segments of transposase, designated patch I and patch II. Second, CY134 on its own has previously been shown to relax the target DNA sequence requirements for Tn10 integration. We provide evidence that suppression by CY134 is not simply a consequence of this mutation conferring a general ``transposition up' phenotype, but rather is due to correcting the original defect. Possible mechanisms of suppression for both CY134 and other second site suppressors are considered.     

The organization of the outside end of transposon Tn5.

The end sequences of the IS50 insertion sequence are known as the outside end (OE) and inside end. These complex ends are related but nonidentical 19-bp sequences that serve as substrates for the activity of the Tn5 transposase. Besides providing the binding site of the transposase, the end sequences of a transposon contain additional types of information necessary for transposition. These additional properties include but are not limited to host protein interaction sites and sites that program synapsis and cleavage events. In order to delineate the properties of the IS50 ends,the base pairs involved in the transposase binding site have been defined. This has been approached through performing a variety of in vitro analyses: a ++hydroxyl radical missing-nucleoside interference experiment, a dimethyl sulfate interference experiment, and an examination of the relative binding affinities of single-site end substitutions. These approaches have led to the conclusion that the transposase binds to two nonsymmetrical regions of the OE, including positions 6 to 9 and 13 to 19. Proper binding occurs along one face of the helix, over two major and minor grooves, and appears to result in a significant bending of the DNA centered approximately 3 bp from the donor DNA-OE junction.     

dnaA, an essential host gene, and Tn5 transposition.

Mutations in dnaA, an essential gene in Escherichia coli, decrease the frequency of transposition of Tn5. An insertion mutation in the dnaA gene does not affect Tn5 gene expression. Therefore, the DnaA protein plays a role either in the transposition reaction itself or in some type of cellular regulation of transposition. Analysis of a mutation in the DnaA box, found at the outside end of IS50, is consistent with a direct interaction of the protein through these bases. IS50 transposition, which utilizes only one end containing a DnaA box, is not affected by dnaA mutations. Overproduction of the DnaA protein does not increase transposition frequencies in wild-type cells, even when the transposase is also overproduced.