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

Williams, Tanya L.; Baker, Tania A.

doi:10.1074/jbc.m308156200

Cited by 8 publications

(9 citation statements)

References 45 publications

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“…Previous studies suggested that MuB allosterically activates MuA. 20,23,38 Our new data extend these studies and support a model in which MuB functions to influence the conformational state of the transpososome in a concerted manner. Thus, we hypothesize that by favoring joined complexes, MuB acts both to stimulate strand transfer and to inhibit disintegration using a common mechanism.…”

Section: Discussionsupporting

confidence: 75%

“…MuB promotes a cooperative change to influence the activity of the Mu transpososome Previous experiments 20,23,26,37 together with the analysis presented above reveal that MuB is a multifaceted activator of MuA and that it is especially important in driving the recombination reaction forward to the final strand transfer complex. To understand how MuB functions to control the activity of the transpososome, we sought to determine if it makes preferential contacts with specific MuA subunits within the transpososome.…”

Section: The Mub Activator Promotes Joining and Antagonizes Reversalmentioning

confidence: 91%

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The Dynamic Mu Transpososome: MuB Activation Prevents Disintegration

Lemberg

Schweidenback

Baker

2007

Journal of Molecular Biology

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DNA transposases use a single active center to sequentially cleave the transposable element DNA and join this DNA to a target site. Recombination requires controlled conformational changes within the transposase to ensure that these chemically distinct steps occur at the right time and place, and that the reaction proceeds in the net forward direction. Mu transposition is catalyzed by a stable complex of MuA transposase bound to paired Mu DNA ends (a transpososome). We find that Mu transpososomes efficiently catalyze disintegration when recombination on one end of the Mu DNA is blocked. The MuB activator protein controls the integration versus disintegration equilibrium. When MuB is present, disintegration occurs slowly and transpososomes that have disintegrated catalyze subsequent rounds of recombination. In the absence of MuB, disintegration goes to completion. These results together with experiments mapping the MuA-MuB contacts during DNA joining suggest that MuB controls progression of recombination by specifically stabilizing a concerted transition to the "joining" configuration of MuA. Thus, we propose that MuB's interaction with the transpososome actively promotes coupled joining of both ends of the element DNA into the same target site and may provide a mechanism to antagonize formation of single-end transposition products.

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Section: Discussionsupporting

confidence: 75%

Section: The Mub Activator Promotes Joining and Antagonizes Reversalmentioning

confidence: 91%

The Dynamic Mu Transpososome: MuB Activation Prevents Disintegration

Lemberg

Schweidenback

Baker

2007

Journal of Molecular Biology

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“…Formation of the Mu transpososome and progression of the complex through the various reaction stages are accompanied by a series of conformational changes resulting in successive increases in transpososome stability (see Mizuuchi and Mizuuchi 2001;Chaconas and Harshey 2002;Goldhaber-Gordon et al 2002a,b;Kobryn et al 2002;Yanagihara and Mizuuchi 2003;Williams and Baker 2004). Some changes in the Mu A monomer structure were apparent upon tetramerization, as noted by comparison (Fig.…”

Section: Catalysis In Transmentioning

confidence: 87%

3D reconstruction of the Mu transposase and the Type 1 transpososome: a structural framework for Mu DNA transposition

Yuan¹,

Beniac²,

Chaconas³

et al. 2005

Genes Dev.

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Mu DNA transposition proceeds through a series of higher-order nucleoprotein complexes called transpososomes. The structural core of the transpososome is a tetramer of the transposase, Mu A, bound to the two transposon ends. High-resolution structural analysis of the intact transposase and the transpososome has not been successful to date. Here we report the structure of Mu A at 16-Å and the Type 1 transpososome at 34-Å resolution, by 3D reconstruction of images obtained by scanning transmission electron microscopy (STEM) at cryo-temperatures. Electron spectroscopic imaging (ESI) of the DNA-phosphorus was performed in conjunction with the structural investigation to derive the path of the DNA through the transpososome and to define the DNA-binding surface in the transposase. Our model of the transpososome fits well with the accumulated biochemical literature for this intricate transposition system, and lays a structural foundation for biochemical function, including catalysis in trans and the complex circuit of macromolecular interactions underlying Mu DNA transposition.[Keywords: DNA transposition; transpososome; transposase; site-specific recombination; 3D reconstruction; electron spectroscopic imaging; scanning transmission electron microscopy] Supplemental material is available at http://www.genesdev.org. A variety of DNA processes including replication, recombination, transcription, and transposition make use of higher-order nucleoprotein complexes (Echols 1986). These complexes are built by multiple proteins binding cooperatively at multiple DNA sites with the assistance of accessory proteins, which bend DNA to facilitate interaction of proteins bound at distant sites. The requirement for the assembly of such elaborate structures ensures high levels of specificity and a means of providing elaborate regulatory mechanisms.Mu DNA was the first mobile element for which an in vitro transposition system (Mizuuchi 1983) was available (for a recent review, see Chaconas and Harshey 2002) and knowledge gained from this system has proven invaluable for more recent analyses of other transposable elements. Interestingly, the Mu system has also turned out to be one of the most complex and elaborate transposition systems studied to date. The Mu strand transfer reaction, the first stage in the transposition process, requires four proteins: Mu A and Mu B encoded by the transposon and HU and IHF from the host cell. Subsequent steps to complete the replicative transposition process of Mu require a variety of host replication proteins.The Mu A protein active site cleaves at the Mu-host junctions and promotes transesterification of the free 3ЈOH Mu ends to a target DNA molecule. An acidic, metal coordinating DDE motif is a crucial element of the Mu active site, as for other transposons (Mizuuchi and Baker 2002). The Mu B protein captures the target DNA, recruits it to the active site of Mu A, and allosterically activates Mu A for the transesterification step. The Escherichia coli HU and IHF proteins are accessory factors that ...

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“…The ATPase activity of MuB is stimulated by DNA and MuA. MuB not only captures target DNA and delivers it to the transpososome, but its interactions with MuA optimize all stages of transpsosome assembly (9, 34–36) (Fig. 4).…”

Section: Transpososome Assembly Activity and Structurementioning

confidence: 99%

“…4A, B) (39, 45, 47). MuB stimulates assembly on both plasmid and oligonucleotide substrates; the stimulation is independent of presence of the FD on oligonucleotide substrates (36). …”

Section: Transpososome Assembly Activity and Structurementioning

confidence: 99%

Transposable Phage Mu

Harshey

2014

Microbiol Spectr

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Transposable phage Mu has played a major role in elucidating the mechanism of movement of mobile DNA elements. The high efficiency of Mu transposition has facilitated a detailed biochemical dissection of the reaction mechanism, as well as of protein and DNA elements that regulate transpososome assembly and function. The deduced phosphotransfer mechanism involves in-line orientation of metal ion-activated hydroxyl groups for nucleophilic attack on reactive diester bonds, a mechanism that appears to be used by all transposable elements examined to date. A crystal structure of the Mu transpososome is available. Mu differs from all other transposable elements in encoding unique adaptations that promote its viral lifestyle. These adaptations include multiple DNA (enhancer, SGS) and protein (MuB, HU, IHF) elements that enable efficient Mu end synapsis, efficient target capture, low target specificity, immunity to transposition near or into itself, and efficient mechanisms for recruiting host repair and replication machineries to resolve transposition intermediates. MuB has multiple functions, including target capture and immunity. The SGS element promotes gyrase-mediated Mu end synapsis, and the enhancer, aided by HU and IHF, participates in directing a unique topological architecture of the Mu synapse. The function of these DNA and protein elements is important during both lysogenic and lytic phases. Enhancer properties have been exploited in the design of mini-Mu vectors for genetic engineering. Mu ends assembled into active transpososomes have been delivered directly into bacterial, yeast, and human genomes, where they integrate efficiently, and may prove useful for gene therapy.

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

Cited by 8 publications

References 45 publications

The Dynamic Mu Transpososome: MuB Activation Prevents Disintegration

The Dynamic Mu Transpososome: MuB Activation Prevents Disintegration

3D reconstruction of the Mu transposase and the Type 1 transpososome: a structural framework for Mu DNA transposition

Transposable Phage Mu

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