Effect of Mutations in the C-terminal Domain of Mu B on DNA Binding and Interactions with Mu A Transposase

Coros, Colin J.; Sekino, Yukiko; Baker, Tania A.; Chaconas, George

doi:10.1074/jbc.m303693200

Cited by 7 publications

(7 citation statements)

References 53 publications

(70 reference statements)

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“…2A and B). The group of G. Chaconas identified a patch of three positively charged lysines as the interacting surface with MuA 11 , 16 . We observed similar results and mapped these residues in the linker region between the N- and C-domains, which in our model occupies an exposed position on the filament surface.…”

Section: Mub Filaments: Some (Dis)assembly Requiredsupporting

confidence: 77%

MuB gives a new twist to target DNA selection

Dramićanin

Ramón‐Maiques

2013

Mobile Genetic Elements

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Transposition target immunity is a phenomenon observed in some DNA transposons that are able to distinguish the host chromosome from their own DNA sequence, thus avoiding self-destructive insertions. The first molecular insight into target selection and immunity mechanisms came from the study of phage Mu transposition, which uses the protein MuB as a barrier to self-insertion. MuB is an ATP-dependent non-specific DNA binding protein that regulates the activity of the MuA transposase and captures target DNA for transposition. However, a detailed mechanistic understanding of MuB functioning was hindered by the poor solubility of the MuB-ATP complexes. Here we comment on the recent discovery that MuB is an AAA+ ATPase that upon ATP binding assembles into helical filaments that coat the DNA. Remarkably, the helical parameters of the MuB filament do not match those of the bound DNA. This intriguing mismatch symmetry led us to propose a model on how MuB targets DNA for transposition, favoring DNA bending and recognition by the transposase at the filament edge. We also speculate on a different protective role of MuB during immunity, where filament stickiness could favor the condensation of the DNA into a compact state that occludes it from the transposase.

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Section: Mub Filaments: Some (Dis)assembly Requiredsupporting

confidence: 77%

MuB gives a new twist to target DNA selection

Dramićanin

Ramón‐Maiques

2013

Mobile Genetic Elements

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“…23,26 How MuA and MuB physically interact during transposition is not well understood, although regions of each protein that are likely to be involved in contacting the other protein have been identified. 23,[27][28][29] In addition, MuB has been suggested to act as an allosteric activator of MuA, and this model is supported and extended by the results of this study.…”

Section: Introductionsupporting

confidence: 81%

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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“…The proteolytic Cterminal fragment of MuB was previously proposed to bind DNA nonspecifically and to interact with MuA through a patch of three lysines, K233, K235, and K236 (25,36). We recognized the Cterminal fragment as an integral part of the AAA+ module, which is connected to the α/β-domain by a linker that harbors the three lysines.…”

Section: Resultsmentioning

confidence: 99%

MuB is an AAA+ ATPase that forms helical filaments to control target selection for DNA transposition

Mizuno

Dramićanin

Mizuuchi

et al. 2013

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

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MuB is an ATP-dependent nonspecific DNA-binding protein that regulates the activity of the MuA transposase and captures target DNA for transposition. Mechanistic understanding of MuB function has previously been hindered by MuB's poor solubility. Here we combine bioinformatic, mutagenic, biochemical, and electron microscopic analyses to unmask the structure and function of MuB. We demonstrate that MuB is an ATPase associated with diverse cellular activities (AAA+ ATPase) and forms ATP-dependent filaments with or without DNA. We also identify critical residues for MuB's ATPase, DNA binding, protein polymerization, and MuA interaction activities. Using single-particle electron microscopy, we show that MuB assembles into a helical filament, which binds the DNA in the axial channel. The helical parameters of the MuB filament do not match those of the coated DNA. Despite this protein-DNA symmetry mismatch, MuB does not deform the DNA duplex. These findings, together with the influence of MuB filament size on strand-transfer efficiency, lead to a model in which MuB-imposed symmetry transiently deforms the DNA at the boundary of the MuB filament and results in a bent DNA favored by MuA for transposition.Phage Mu | nucleoprotein filament D NA transposons are ubiquitous in the genomes of all forms of life and play important evolutionary roles in generating gene diversity and in shaping genomic landscapes (1). Although typically transposons exhibit no strong sequence selectivity for the target DNA site, certain transposons avoid self-destructive insertion (reviewed in ref.2), a phenomenon called "target immunity" because the presence of a copy of the transposon renders nearby DNA sites "immune" to additional insertion by the same transposon (3-8). MuB plays critical roles in this selfimmunity in the bacteriophage Mu transposition process.Phage Mu is one of the most complex and efficient transposable elements (reviewed in refs. 3 and 4). Two phage-coded proteins, MuA and MuB, are essential for efficient Mu transposition. MuA is the transposase responsible for synapsing the two Mu end sequences and for all of the DNA cutting and joining steps in the initial stages of transposition. However, transposition is inefficient in the absence of MuB, and the residual Mu insertion that takes place uses only DNA target sites near or within the transposing element, often leading to self-destruction (9-11). MuB is a small (35-kDa) ATP-dependent nonspecific DNA-binding protein with relatively low ATPase activity (10,12,13). Upon ATP binding, MuB polymerizes preferentially on DNA, but in the absence of DNA it still can form polymers of variable sizes (14, 15). When observed by total internal reflection fluorescence microscopy, GFP-MuB-ATP binds along the DNA molecule forming many short separate segments of polymers, and, as more GFP-MuB is added, the protein-covered segments elongate to form an apparently continuous polymer that fully coats the DNA. Hydrolysis of ATP reverses this process, triggering disassembly of the MuB polymer (16-18)...

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Effect of Mutations in the C-terminal Domain of Mu B on DNA Binding and Interactions with Mu A Transposase

Cited by 7 publications

References 53 publications

MuB gives a new twist to target DNA selection

MuB gives a new twist to target DNA selection

The Dynamic Mu Transpososome: MuB Activation Prevents Disintegration

MuB is an AAA+ ATPase that forms helical filaments to control target selection for DNA transposition

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