Site-specific recombination by the DDE family member mobile element IS30transposase

Abstract

DNA rearrangements carried out by site-specific recombinases and transposases (Tpases) show striking similarities despite the wide spectrum of the catalytic mechanisms involved in the reactions. Here, we show that the bacterial insertion sequence (IS)30 element can act similarly to site-specific systems. We have developed an inversion system using IS30 Tpase and a viable phage, where the integration͞excision system is replaced with IS30. Both models have been proved to operate analogously to their natural counterpart, confirming that a DDE family Tpase is able to fulfill the functions of site-specific recombinases. This work demonstrates that distinction between transposition and site-specific recombination becomes blurred, because both functions can be fulfilled by the same enzyme, and both types of rearrangements can be achieved by the same catalytic mechanisms.I t is now well documented that genetic information can be reshuffled by mobile elements that are usually distinguished as site-specific systems and transposons. Even though the output of their activity can be simplified to insertion, deletion, or inversion of DNA segments, transposition and site-specific recombination are generally regarded as distinct phenomena (1). Classification according to the protein-sequence motifs revealed that some transposases (Tpases) and site-specific recombinases (Recases) with entirely different functions fall within the same family (2-6). This finding supports that mechanistically dissimilar enzymes can perform similar biological functions and suggests that transposition and site-specific recombination may be closer to each other in some respect than was supposed earlier. Further support for this idea may emerge from insertion sequence (IS) elements that form an active junction composed of the inverted repeats (IRs) (7-10). This IR-IR junction shows similarities in its structure and function to the recombination sites of sitespecific systems.The Escherichia coli element, IS30, encoding a Tpase with the well conserved DDE signature (11), belongs to the increasing class of elements that transpose through an intermediate formed by abutted IRs (8). The IR-IR joint bracketing a 2-bp spacer is composed of the 26-bp left and right IR ends (IRL and IRR, respectively) that differ at three positions (12). IR-IR joints occur in both IS dimers and minicircles that can integrate into hot spot sequences or next to other IS30 ends (13-15). Whereas transposition into a hot spot is a conventional way of translocation, which accompanies the resolution of IR-IR junction, targeting an IR end seems more specific for IS30 and generates a new IR-IR joint. The IRs, together with the adjacent IS sequences, are important for the directionality of recombination, because, as always, IRL-IRR junction arises through dimerization or targeting an IR (8, 13, 16). The most frequent reaction has been observed when both donor and target DNA contain an IR-IR junction (13). In this case, the rearrangement is remarkably reversible and resembles site-specific ...

Characterization of the Variants, Flanking Genes, and Promoter Activity of the Leifsonia xyli subsp. cynodontis Insertion Sequence IS 1237

“…Furthermore, we noticed that the putative transposase of IS1237 contained the DDE motif (23). Recent research confirmed that a DDE family transposase is able to perform the functions of site-specific recombinases (22). Thus, it is possible that this transposase functions as a recombinase to catalyze the site-specific deletion of IS1237 in L. xyli subsp.…”

Evolutionary Dynamics of Insertion Sequences in Helicobacter pylori

“…Chromosomal DNA was prepared from confluent cultures on brain heart infusion agar plates either by the hexadecyltrimethylammonium bromide extraction method as described previously (39) or with a QIAamp DNA minikit (QIAGEN Inc., Chatsworth, Calif.). Primers used for generating probes for hybridization, PCR, and nucleotide sequencing are listed in Table 1.…”

“…The orfA genes of IS605 and IS607 encode transposases belonging to two different families. IS607 orfA is predicted to encode a serine recombinase, most homologs of which catalyze only site-specific recombination (59), although one case in which this enzyme mediates both site-specific recombination and transposition has also been reported (39). In contrast, the IS605 orfA product is not considered to be a serine recombinase based on amino acid homologies.…”

Everyman's Guide to Bacterial Insertion Sequences

“…The founding member of the family, IS30, is the best characterized at the mechanistic level (117,(182)(183)(184)(185)(186)(187)(188)(189) and an in vitro transposition system has been developed (190). This 1,221 bp long Escherichia coli element belongs to a growing class of IS known to transpose through an intermediate formed by abutting the IR, donor primed transposon replication.…”