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Genetic Analysis of the Interaction of the Insertion Sequence IS903 Transposase with Its Terminal Inverted Repeats

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Genetic Analysis of the Interaction of the Insertion Sequence IS903 Transposase with Its Terminal Inverted Repeats Proc. Nail. Acad. Sci. USA Vol. 84, pp. 8049-8053, November 1987 Genetics Genetic analysis of the interaction of the insertion sequence IS903 transposase with its terminal inverted repeats (point mutations/cis-acting protein/DNA-protein interactions/transposition mechanisms) KEITH M. DERBYSHIRE, LEON HWANG, AND NIGEL D. F. GRINDLEY Yale University, Department of Molecular Biophysics and Biochemistry, New Haven, CT 06510 Communicated by Joan A. Steitz, August 10, 1987 (receivedfor review June 26, 1987) ABSTRACT The insertion sequence IS903 has perfect, kanamycin (7, 8). Each IS903 element is capable of transpo- 18-base-pair terminal repeats that are the presumed binding sition and is flanked by perfect 18-bp inverted repeats. These sites of its transposase. We have isolated mutations throughout are the only sequence elements required in cis for transpo- this inverted repeat and analyzed their effect on transposition. sition (9), although it should also be noted that the IS903 We show that every position in the inverted repeat (with the transposase is a cis-acting protein (7). This is a phenomenon possible exception of position 4) is important for efficient that appears to be a general property of IS element trans- transposition. Furthermore, various substitutions at a single posases; those of IS], ISIO, and IS50 also do not efficiently position can have a wide range of effects. Analysis of these complement mutant transposons in trans (10-12). We have hierarchical effects suggests that transposase contacts the described (13) a saturation mutagenesis technique by which minor groove in the region from position 13 to position 16 but we isolated many mutations throughout the inverted repeat of makes major groove (or more complex) interactions with the IS903. We have used these to characterize the interaction of outer portion of the inverted repeat. Our data indicate that the the IS903 transposase with its inverted repeats. transposase exhibits relaxed specificity for the "second" end of a transposed segment; the defect in transposition ofvirtually all MATERIALS mutant inverted repeats can be rescued by a wild-type end. AND METHODS However, this rescue exhibits a pronounced position effect; in Standard molecular and genetic techniques were used (14). most cases, it is efficient only when the wild-type end is close to DNA sequences were determined by the dideoxynucleotide the 3' end of the transposase gene. This confirms the cis-acting chain-termination procedure (15, 16). All point mutations, nature of the transposase protein and suggests the initial with the exceptions described below, were isolated as de- transposase-inverted repeat interaction is the rate-limiting step scribed (13). Point mutations at the thymidines at positions 11 in transposition. From the behavior of transposons with one and 12 (T1' and T12) were isolated by the oligonucleotide- mutant and one wild-type end, we infer that the inverted repeat directed mutagenesis procedure of Kunkel (17). Oligonucle- contains two functional domains-one for initial complex otides for mutagenesis and sequencing were made on a model formation with transposase and the other for effective comple- 8600 synthesizer from Biosearch (San Rafael, CA). tion of transpositional recombination. To support this hypoth- Plasmids. The vector used for cloning the mutant ends esis we show that an end with a mutation in one domain can pKD50 (Fig. 1) is a derivative of pBR322 that has been significantly rescue an end with a mutation in the other domain. modified in the following ways. (i) A 1.5-kilobase (kb) fragment encoding chloramphenicol resistance (9) has been One of the striking characteristics of most bacterial trans- cloned into the Pvu II site. (ii) An Xba I linker has been posons is that they have terminal inverted repeats. These inserted into the Sal I site. (iii) A Sal I linker has been inserted repeats define the transposing segment since they are joined into the EcoRI site (the original Pvu II, Sal I, and EcoRI sites precisely to the target site in transposition, and they must be were all destroyed in these steps). (iv) A segment of DNA correctly oriented for transposition to occur. It is assumed encoding the IS903 transposase expressed from the lacUV5 that these sequences are the recognition sites of the element- promoter (18) has been cloned between the new Sal I site and encoded transposase, because it has been clearly demon- the BamHI site of pBR322. IS903 ends were cloned into strated, for many transposons, that these are the only DNA pKD50 to generate model transposons as follows. Single- sequences required in cis for efficient transposition. The stranded DNA from the M13 clones was made double inverted repeats ofdifferent insertion sequences (ISs) vary in stranded by primer extension, divided into two aliquots, and length from 8 base pairs (bp) (IS91) to 40 bp (IS2) and in their then digested with either BamHI and Pst I or with Xba I and degree of homology (1-4). Although the inverted repeats of HindIII. After heat treatment (65°C, 10 min), the two reaction many insertion elements have been defined by sequence and mixtures were mixed with the 6.2-kb BamHI-Xba I fragment deletion analysis, a comprehensive analysis of the effects of from pKD50 and a 1-kb Pst I-HindIII fragment containing the mutations in an inverted repeat has not been carried out. A kanamycin resistance gene of Tn903 [the HindIII site nor- single-base transition at the outside end of IS50 reduces mally present within the kanamycin resistance gene has been transposition by a factor of 100 (5). Three point mutations in destroyed by bisulfite mutagenesis (19)]. Ligation yielded the outer 13 bp of TnlO also reduce transposition by a factor pKD100 (with wild-type transposon ends) and analogous of 100 (6). We, therefore, decided to analyze the effects of plasmids each with two copies ofa single mutant end (Fig. 1). single substitutions in the inverted repeat of IS903. All inverted repeats cloned into pKD50 have been checked Tn903 is a composite transposon consisting of two identi- by restriction endonuclease and sequence analysis. cal, 1057-bp, IS903 elements that lie in inverted orientation Plasmids with a similar structure to pKD100 were made in on either side of a DNA segment encoding resistance to which the transposon had hybrid ends-one wild-type and one mutant (see Fig. 1). These plasmids contain a 2.5-kb Pst The publication costs of this article were defrayed in part by page charge I fragment carrying the Escherichia coli galK gene inserted payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: IS, insertion sequence. 8049 8050 Genetics: Derbyshire et al. Proc. Natl. Acad. Sci. USA 84 (1987) A from these pOX38: :Tn derivatives, the majority ofthe pOX38 INVERTED REPEAT GATCCTCTAGABarn Xba ______________ Pat Hin DNA was deleted. The DNA was digested to completion with GGATCCTCTAGA GATITATC CAGC CTGCAGAAGC1T Cla I (which cuts only outside the transposon and at least seven times within pOX38) and religated after diluting the DNA. Chloramphenicol-resistant transformants were select- B ed, and these contained smaller, high copy number plasmids containing the entire transposon. This DNA was used direct- ly for sequence analysis (16) using primers that enabled each S B x P S inverted repeat-target junction to be sequenced. pKD50 _ n pI Tnpase -_ Cm Ori APr RESULTS S BX P H PX PS Point Mutations Throughout the Inverted Repeat Affect pKD100 I----rL S IS903 Transposition. It has been suggested (9) that the outer Tnpase-* IRp Km' IR Cmr Ori Apr 20 bp ofIS903 were the only sequences necessary for efficient P d transposition. To confirm this we have made two trans- posase-defective derivatives of pKD100. One has a frame- shift mutation at the Mlu I site early in the gene; the other has Hybrid M end a deletion ofthe entire segment carrying the transposase gene elements and its promoter (the Sal I-BamHI fragment in Fig. 1). When +M I., - -- ------I complemented in trans by a copy of the lacUV5-promoted M transposase gene on pACYC184 both derivatives gave trans- positions of chloramphenicol resistance at the same frequen- cy (0.5%-1% ofthe transposition frequency ofpKD100). This pLH7 L lI confirms the strong preference of the IS903 transposase for + GaIK acting in cis and shows that the 18-bp terminal inverted repeats are the only cis-acting sequences required for trans- position. pK1D91 L---l U Each of the mutant inverted repeats was subcloned from M13mplO into the tester plasmid to reconstruct a transposon FIG. 1. Construction of IS903 elements with mutant ends. (A) with two copies ofthe same mutant end and was then assayed Sequence of the wild-type inverted repeat and its location in the for transposition (Fig. 2). It is clear that every base pair within polylinker of M13mplO. Mutant inverted repeats were subcloned the inverted repeat (with the possible exception ofposition 4) from M13 into pKD50. (B) The basic structures of pKD50, pKD100, is important for efficient transposition. However, different and other constructs are shown, along with relevant restriction sites. substitutions at a single position may exhibit substantially pKD100 has two wild-type inverted repeats (IR) that are located different effects (the hierarchy of nucleotide substitutions is proximal (IRp) and distal (IRd) to the transposase gene. The hybrid- shown at the relevant positions). One of the more striking ended elements show the two locations used for the mutant (M) and effects is seen at the thymidine at position 11 where a change wild-type (+) ends. These plasmids also contain the galK gene on a to a guanosine (T1--+G) has only a small effect on transpo- 2.5-kb Pst I fragment inserted in the Pst I site of the ampicillin adenosine or cytidine resistance gene.
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