Control of transposase activity within a transpososome by the configuration of the flanking DNA segment of the transposon

Mizuuchi, Michiyo; Rice, Phoebe A.; Wardle, Simon J.; Haniford, David B.; Mizuuchi, Kiyoshi

doi:10.1073/pnas.0706556104

Cited by 7 publications

(8 citation statements)

References 40 publications

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“…2B). The disintegration reaction was also observed for Mu, on both oligonucleotide and plasmid substrates, and required high temperatures or altered metal ions (29, 30). Both true as well as pseudo reversal were observed for Mu (Fig.…”

Section: Disintegrationmentioning

confidence: 91%

“…Ca 2+ ions, which support ST of precleaved ends, also supported true reversal; these ions did not support pseudo reversal, suggesting that the metal ion binding pocket is similar in the forward and the true-reversal reactions, but different in the foldback reaction. When the transpososome was assembled on uncleaved Mu donor substrates, and the reaction proceeded through 3’ Mu end cleavage and ST, true reversal required high temperatures (29, 30), suggesting that in the normal course of events, the reactive groups are rearranged within the ST complex such that reversal is proscribed; high temperatures likely cause conformation changes that restore the pre-ST configuration of the active site (29). The higher stability of the ST complex compared to the cleaved complex (31), is consistent with the notion of structural transitions in the transpososomes (and hence the active sites) as the reaction proceeds forward.…”

Section: Disintegrationmentioning

confidence: 99%

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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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Section: Disintegrationmentioning

confidence: 91%

Section: Disintegrationmentioning

confidence: 99%

Transposable Phage Mu

Harshey

2014

Microbiol Spectr

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“…This contrasts with assembly of Mu transposase on DNA substrate mimicking the transposition product, where multiple species were inferred. 14 The structure of the PFV intasome and STC remains the only high-resolution structures of a retroviral integrase in complex with DNA substrate. Although the structures of the analogous HIV-1 complexes are likely to be similar and can be modeled on the PFV structure, 15 especially in the immediate vicinity of the active site, the structure of the HIV-1 complexes are required to fully understand the mechanism of integrase inhibitors and the mutations that confer drug resistance.…”

Section: Discussionmentioning

confidence: 99%

Assembly of prototype foamy virus strand transfer complexes on product DNA bypassing catalysis of integration

et al. 2012

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Integrase is the key enzyme that mediates integration of retroviral DNA into cellular DNA which is essential for viral replication. Inhibitors of HIV-1 that target integrase recognize the nucleoprotein complexes formed by integrase and viral DNA substrate (intasomes) rather than the free enzyme. Atomic resolution structures of HIV-1 intasomes are therefore required to understand the mechanisms of inhibition and drug resistance. To date, prototype foamy virus (PFV) is the only retrovirus for which such structures have been determined. We show that PFV strand transfer complexes (STC) can be assembled on product DNA without going through the normal forward reaction pathway. The finding that a retroviral STC can be assembled in this way may provide a powerful tool to alleviate the obstacles that impede structural studies of nucleoprotein intermediates in HIV-1 DNA integration.

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“…After strand transfer the complex is so stable that the “enzyme” MuA does not actually turn over. The strand transfer reaction can only be reversed if the complex is disrupted, for instance by heating to 75C 12,13 . This may be a thermodynamic necessity for a reaction in which the 1 st step (hydrolysis) is committed, yet the 2 nd step (strand transfer) is chemically isoenergetic, with no net change in the number of phosphodiester bonds.…”

mentioning

confidence: 99%

The Mu transpososome structure sheds light on DDE recombinase evolution

Montaño

Pigli

Rice

2012

Nature

Self Cite

138

197

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Studies of bacteriophage Mu transposition paved the way for understanding retroviral integration and V(D)J recombination as well as many other DNA transposition reactions. Here we report the structure of Mu transposase (MuA) in complex with bacteriophage DNA ends and target DNA, determined from data that extend anisotropically to 5.2/5.2/3.7Å resolution, in conjunction with previously-determined structures of individual domains. The highly intertwined structure illustrates why chemical activity depends on formation of the synaptic complex, and reveals that individual domains play different roles when bound to different sites. It also suggests explanations for the increased stability of the final product complex and for its preferential recognition by the ATP-dependent unfoldase ClpX. Although MuA and many other recombinases share a structurally conserved “DDE” catalytic domain, comparisons among the limited set of available complex structures suggest that some conserved features, such as catalysis in trans and target DNA bending, arose through convergent evolution because they are important for function.

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Control of transposase activity within a transpososome by the configuration of the flanking DNA segment of the transposon

Cited by 7 publications

References 40 publications

Transposable Phage Mu

Transposable Phage Mu

Assembly of prototype foamy virus strand transfer complexes on product DNA bypassing catalysis of integration

The Mu transpososome structure sheds light on DDE recombinase evolution

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