The Tn7 transposase is a heteromeric complex in which DNA breakage and joining activities are distributed between different gene products.

Sarnovsky, Robert; May, Earl W.; Craig, Nancy L.

doi:10.1002/j.1460-2075.1996.tb01024.x

Cited by 145 publications

(159 citation statements)

References 73 publications

(139 reference statements)

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“…Activators of transposition such as MuB have not been generally found for cut-and-paste transposases that promote recombination of a transposon end through the repeated use of the same active site. (The cut-and-paste transposition system of Tn7 employs a MuB-like protein, although active site residues from two different protein subunits are required to recombine a single transposon DNA end (43,44)). Cut-and-paste transposition severs the connection between both strands of the flanking DNA and the transposon end before the joining step.…”

Section: Discussionmentioning

confidence: 99%

Reorganization of the Mu Transpososome Active Sites during a Cooperative Transition between DNA Cleavage and Joining

Williams¹,

Baker

2004

Journal of Biological Chemistry

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Transposition of mobile genetic elements proceeds through a series of DNA phosphoryl transfer reactions, with multiple reaction steps catalyzed by the same set of active site residues. Mu transposase repeatedly utilizes the same active site DDE residues to cleave and join a single DNA strand at each transposon end to a new, distant DNA location (the target DNA). To better understand how DNA is manipulated within the Mu transposase-DNA complex during recombination, the impact of the DNA immediately adjacent to the Mu DNA ends (the flanking DNA) on the progress of transposition was investigated. We show that, in the absence of the MuB activator, the 3 -flanking strand can slow one or more steps between DNA cleavage and joining. The presence of this flanking DNA strand in just one active site slows the joining step in both active sites. Further evidence suggests that this slow step is not due to a change in the affinity of the transpososome for the target DNA. Finally, we demonstrate that MuB activates transposition by stimulating the reaction step between cleavage and joining that is otherwise slowed by this flanking DNA strand. Based on these results, we propose that the 3 -flanking DNA strand must be removed from, or shifted within, both active sites after the cleavage step; this movement is coupled to a conformational change within the transpososome that properly positions the target DNA simultaneously within both active sites and thereby permits joining.The successful relocation of mobile genetic elements, as with all DNA rearrangement processes, requires the precise spatial and temporal organization of DNA components within the active sites of large nucleoprotein complexes. This dynamic organization permits the proper series of DNA cleavage and joining reactions that comprise each DNA recombination pathway. Transposition and retroviral integration occur via one of two pathways that share common reaction steps (1, 2). Similar phosphoryl transfer reactions also take place during the early steps of VDJ recombination (3). Many transposases and integrases form a recombinase family related both by the threedimensional structure of their catalytic domains and by a conserved set of acidic active site residues (4). Bacteriophage Mu transposase is a well studied member of this family.Transposases and integrases promote recombination via either a replicative or cut-and-paste transposition pathway. These pathways share two common steps (see Fig. 1). First, one strand at each element end is hydrolyzed to yield a 3ЈOH at the terminus of the element sequence. This donor cleavage step separates this strand at each element end from the surrounding (or flanking) DNA. In the second common step, called DNA strand transfer or joining, each liberated 3ЈOH directly attacks closely spaced phosphodiester bonds in opposite strands of the DNA at the new location (the target DNA). Mu transposase promotes these two steps during replicative transposition to yield a transposition product in which each transposon end is covalently attached ...

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Section: Discussionmentioning

confidence: 99%

Reorganization of the Mu Transpososome Active Sites during a Cooperative Transition between DNA Cleavage and Joining

Williams¹,

Baker

2004

Journal of Biological Chemistry

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“…1) in which the element is first excised from the donor site by double-strand breaks at each end of the transposon, followed by the joining of these transposon ends to the target DNA. These DNA breakage and joining activities are executed by TnsA and TnsB, which collaborate to form the transposase (15,16). TnsC plays a key role in the regulation of transposition and in target site selection.…”

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Recognition of triple-helical DNA structures by transposon Tn7

Rao

Miller

Craig

2000

Proc. Natl. Acad. Sci. U.S.A.

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We have found that the bacterial transposon Tn7 can recognize and preferentially insert adjacent to triple-helical nucleic acid structures. Both synthetic intermolecular triplexes, formed through the pairing of a short triplex-forming oligonucleotide on a plasmid DNA, and naturally occurring mirror repeat sequences known to form intramolecular triplexes or H-form DNA are preferential targets for Tn7 insertion in vitro. This target site selectivity depends upon the recognition of the triplex region by a Tn7-encoded ATP-using protein, TnsC, which controls Tn7 target site selection: the interaction of TnsC with the triplex region results in recruitment and activation of the Tn7 transposase. Recognition of a nucleic acid structural motif provides both new information into the factors that influence Tn7's target site selection and broadens its targeting capabilities.

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“…TnsB binds specifically to multiple sites within the ends of Tn7 (8, 9) and mediates 3′ end breakage and joining (5), whereas TnsA mediates 5′ end breakage (4). Because TnsA does not bind specifically to DNA (10,11), and no TnsB end breakage is seen in the absence of TnsA (12), we have proposed that interaction of TnsA with TnsB recruits TnsA to the Tn7 ends and activates TnsB (10,12).…”

mentioning

confidence: 99%

“…Many transposases contain a single type of polypeptide (1), but the Tn7 transposase is heteromeric, containing two polypeptides, TnsA and TnsB (4,5). Hundreds of elements containing TnsA-like and TnsB-like genes are present in bacterial genomes (6,7).…”

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confidence: 99%

Direct interaction between the TnsA and TnsB subunits controls the heteromeric Tn7 transposase

Choi

Liu

Sarnovsky

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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The transposon Tn7 transposase that recognizes the transposon ends and mediates breakage and joining is heteromeric. It contains the Tn7-encoded proteins TnsB, which binds specifically to the transposon ends and carries out breakage and joining at the 3′ ends, and TnsA, which carries out breakage at the 5′ ends of Tn7. TnsA apparently does not bind specifically to DNA, and we have hypothesized that it is recruited to the ends by interaction with TnsB. In this work, we show that TnsA and TnsB interact directly and identify several TnsA and TnsB amino acids involved in this interaction. We also show that TnsA can stimulate two key activities of TnsB, specific binding to the ends and pairing of the Tn7 ends. The ends of Tn7 are structurally asymmetric (i.e., contain different numbers of TnsB-binding sites), and Tn7 also is functionally asymmetric, inserting into its specific target site, attachment site attTn7 (attTn7) in a single orientation. Moreover, Tn7 elements containing two Tn7 right ends can transpose, but elements with two Tn7 left ends cannot. We show here that TnsA + TnsB are unable to pair the ends of a Tn7 element containing two Tn7 left ends. This pairing defect likely contributes to the inability of Tn7 elements with two Tn7 left ends to transpose.

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The Tn7 transposase is a heteromeric complex in which DNA breakage and joining activities are distributed between different gene products.

Cited by 145 publications

References 73 publications

Reorganization of the Mu Transpososome Active Sites during a Cooperative Transition between DNA Cleavage and Joining

Reorganization of the Mu Transpososome Active Sites during a Cooperative Transition between DNA Cleavage and Joining

Recognition of triple-helical DNA structures by transposon Tn7

Direct interaction between the TnsA and TnsB subunits controls the heteromeric Tn7 transposase

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