Transposons transposase

A strain Mycobacterium sp. MR65 was developed by cloning the dszABCD genes from R. erythropolis KA2-5-1 into a Mycobacterium sp. NCIMB 10403 strain via a transposon-transposase complex [228],... 

Transposons are mobile DNA elements (sizes 2.5-23 kbp) that move from one place to another in the chromosome or onto extrachromosomal genetic elements within the same cell. They are flanked by inverted repeats at then-ends and encode among other proteins a transposase that is needed for the transposition process. Resistance genes in the transposon are often parts of integrons. These are structures that cany an integrase responsible for the insertion of the resistance gene cassettes into the integron. 

Many examples of mobile elements are found in bacteria, where they are called transpo-sons. Bacterial transposons have terminal repeat sequences that both code for the enzymes catalyzing the process of transposition (transposases) and physically interact with these enzymes to bring them to the DNA target site. At this site the DNA-bound transposase presumably catalyzes the endonucleolytic cleavage of the terminal repeat sequence of the trahsposon and also catalyzes a similar sequence in the target DNA. 

Bacteria have two classes of transposons. Insertion sequences (simple transposons) contain only the sequences required for transposition and the genes for proteins (transposases) that promote the process. Complex transposons contain one or more genes in addition to those needed for transposition. These extra genes might, for example, confer resistance to antibiotics and thus enhance the survival chances of the host cell. The spread of antibiotic-resistance elements among disease-causing bacterial populations that is rendering some antibiotics ineffectual (pp. 925-926) is mediated in part by transposition. 

Bacterial transposons vary in structure, but most have short repeated sequences at each end that serve as binding sites for the transposase. When transposition occurs, a short sequence at the target site (5 to 10 bp) is duplicated to form an additional short repeated sequence that flanks each end of the inserted transposon (Fig. 25-42). These duplicated segments result from the cutting mechanism used to insert a transposon into the DNA at a new location. 

A second Drosophila transposon called mariner630 typifies the mariner / Tel transposon superfamily, which also contains members from nematodes,631 other invertebrates, fishes,632 amphibia,633 and possibly human beings.634 These transposons encode a transposase containing a D, D, D or D, D, E motif630 but no other proteins. They contain short 30-bp terminal inverted repeats and become inserted into host TA sequences.631 Movement of some repetitive sequences of the LINE635 and SINE636 families within the human genome may be assisted by mariner transposons.637... 

Many DDE transposases carry a DNA sequence-specific binding domain in their N-terminal regions and at least one domain involved in multimerization. Generally, the catalytic domain is located in the C-terminal part of the protein. The DDE domain is by far the most studied and best understood catalytic motif involved in transposition. It is found in many different types of transposon from retroviruses to Tc-Mariner, bacterial ISs, and transposons (4). 

Another regulatory mechanism, called trans-cleavage, is considered a quality control of the transposition reaction. Transposition requires the formation of a specific complex between the transposase and both transposon ends called a trans-pososome. In transcleavage regnlation, transposase bound at one end is constrained to cleave the opposite transposon end. This obliges prior formation of the transpososome before DNA strand... 

For eukaryotic transposons, assembly of a transpososome is also required for transposition. The Hermes transposase is active as a hexamer on DNA (10) and Himarl transposase is active as a tetramer (11), although it remains unclear whether the Mosl transposase is active as a dimer or a tetramer (12, 13). For the well-described P Element, the transposase is active as a tetramer, and it has been reported recently that GTP acts as an allosteric cofactor for synapsis (14). 

Although all transposons with a DDE transposase ( DDE transposons ) use this type of chemistry, a large diversity exists in the overall transposition mechanism. As explained above, DDE transposases catalyze only single-strand cleavage and transfer of the 3 -OH transposon ends (the transferred strand). However, to liberate the transposable element from donor DNA, the transposase must deal with the second DNA strand (also called the nontransferred strand (3, 24). A subclassification of DDE transposons is based on the mechanisms used to manage this. 

For the widely dispersed Tc-Mariner transposon group, the transposase first cleaves within the 5 end of the transposon—the nontransferred strand. This activity resembles the nuclease activity (see above) that simply terminates at the cleavage step and does not take in charge the strand transfer step. Moreover, unlike transposition reactions, this cleavage does not require formation of a synaptic complex (29). The transferred strand is cleaved at the very tip of the TE. The fact that the nontransferred strand is cleaved within the transposon results in retention of the few TE-speciflc bases in the donor molecule after TE excision. After resealing and repair, the donor backbone retains several additional base pairs derived from the TE (called a scar) that marks the passage of the transposon (2). 

Interestingly, transposon Tn7 behaves in a similar way but, in this case, the 5 endonuclease activity is supplied by a separate enzyme whose structure resembles that of a type II restriction enzyme (30), and cleavage occurs cleanly at the transposon tip rather than within the TE. Transposition of Tn7, like most bacterial elements, does not leave a scar. In both the Tc-Mariner transposon group and the Tn7 family of transposons, the transposase then cleaves and transfers the 3 end in a true DDE transposition reaction (Fig. le). 

Although the reactions catalyzed by the S- and Y- site-specific recombinases are weU characterized, little is known abont the biochemistry of their cousins, the Y- and S-transposases. These transposases are thonght to catalyze strand breakage and transfer as do their site-specific recombinase relatives. Neither of these requires divalent metal ions for catalysis. Althongh these transposases show site-specificity for the ends of the donor transposon, they seem more flexible than classic site-specific... 

S. aureus transposons are small mobile elements that often encode resistance genes (P-lactamase, resistance to erythromycin and tetracycline). All the transposons encode a transposase gene, and the product of this gene catalyses excision and/or replication of the element, as well as integration. Horizontal transfer of transposons to other S. aureus cells is presumably mediated by another MGE that is transferred, most likely a plasmid transferred by transduction or conjugation. Conjugative transposons have also been described in S. aureus. However, it is not clear if native conjugative transposons are found in S. aureus (Novick 1990). 

Figure 1. Map of the transposon Tn5. The insertion sequences IS50L and IS50R flank the central region which contains the genes for resistance to kanamycin (Km ), bleomycin (Bler), and streptomycin (Sm ). The P refers to the position of the promoters for expression of antibiotic resistance, transposase and transpoase inhibitor (on IS50R). The restriction sites are marked X=XhoI, B=BgIII, and H=HindIII.

Several bacterial transposons, referred to as insertion elements (ISelements), consist only of a gene that codes for a transposition enzyme (i.e., transposase), flanked by short DNA segments called inverted repeats (Figure 18.16). (Inverted repeats are short palindromes.) More complicated bacterial transposable elements, called composite transposons, contain additional genes, several of which may code for antibiotic resistance. Because transposons can jump between bacterial chromosomes, plasmids, and viral genomes, transpositions are now believed to play an important role in the spread of antibiotic resistance among bacteria. 

Transposase-mediated integration, generation of a transposon library... 

Since McClintock s early work on mobile elements in corn, transposons have been identified in other eukaryotes. For Instance, approximately half of all the spontaneous mutations observed in Drosophila are due to the Insertion of mobile elements. Although most of the mobile elements in Drosophila function as retrotransposons, at least one—the P element—functions as a DNA transposon, moving by a cut-and-paste mechanism similar to that used by bacterial insertion sequences. Current methods for constructing transgenic Drosophila depend on engineered, high-level expression of the P-element transposase and use of the P-element Inverted terminal repeats as targets for transposition. 

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