The solution structure of the C-terminal domain of the Mu B transposition protein

Hung, Ling‐Hong; Chaconas, George; Shaw, Gary S.

doi:10.1093/emboj/19.21.5625

Cited by 10 publications

(16 citation statements)

References 70 publications

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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%

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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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Section: Resultsmentioning

confidence: 99%

“…termined by NMR spectroscopy and reported to resemble the Nterminal domain of Escherichia coli replicative helicase DnaB (25) (Fig. S1) In this study we combine bioinformatic, mutagenic, biochemical, and electron microscopy (EM) techniques to characterize the structure of MuB and its role in DNA binding and transposition.…”

mentioning

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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show abstract

“…Mutagenesis and purification of the Mu B 223-312 gene was performed using the Mu B 223-312 gene expression construct, pHH05 from the strain GC1876, described previously (40). One liter of cells was grown in LB in a 4-liter Fernbach flask in the presence of 50 g/ml ampicillin and 1% (w/v) glucose at 37°C, with a rotation of 225 rpm, to an A 595 of 0.65.…”

Section: Methodsmentioning

confidence: 99%

“…It was proposed that this patch may be involved in the DNA binding activity that has been associated with the C-terminal domain. In this study, we first confirmed that these residues are responsible for binding DNA, and then we investigated how these positively charged residues contribute to the in vitro The ribbon diagram, generated with the Swiss Pdb Viewer, represents the average of the 20 defined solution structures previously determined from NMR data with the initial 8 residues removed (40). The four helixes have been colored in green, blue, yellow, and purple and labeled as helix 1, 2, 3, and 4 respectively.…”

mentioning

confidence: 87%