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

Abstract

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)...

Unlocking Tn3-family transposase activity in vitro unveils an asymetric pathway for transposome assembly

“…For Mu, the target adaptor MuB binds DNA with relatively low specificity, preferring A/T-rich DNA stretches (23,24,40). In contrast, TnsC is addressed to specific sites by the Tn7-encoded target selector proteins TnsD or TnsE.…”

“…In addition to mediating transposition, TnpA is responsible for target immunity, a long-range regulatory phenomenon whereby transposons avoid inserting more than once into the same DNA region (18,19). Other systems with target immunity, such as phage Mu or Tn7, require an adaptor protein (MuB and TnsC, respectively) that controls integration by directing the transpososome to appropriate targets (20)(21)(22)(23)(24). The Tn3-family TnpA is the only transposonencoded protein directly involved in immunity and is the only possible determinant to account for the specificity whereby each element confers immunity to itself but not to other family members (18,25).…”

Transposition of insertion sequence IS<i>256Bsu1</i> in <i>Bacillus subtilis</i> 168 is strictly dependent on <i>recA</i>

“…There is a 12-aa insertion in RecA between the two domains corresponding to the N-terminal appendage and α/β domain of MuB, and also a 16-aa insertion in the homologous loop L1 region. Mutations at basic residues in the L1 region of MuB cause MuA to lose responsive ATPase activity (Mizuno et al, 2013). According to their corresponding residues in the higher structure of MuB, R58 of B. subtilis RecA resides in the N-terminal appendage and E154, G155, D159 and G202 are within the α/β domain of the AAA + module.…”

“…In particular, G202 of B. subtilis RecA is in the corresponding Walker B motif within the α/β domain of MuB, while K70, F215, K241 and K243 of RecA have no homologous counterpart in MuB although the flanking regions are conserved. The N-terminal appendage of MuB has nothing to do with ATP hydrolysis, interaction with MuA or DNA binding (Mizuno et al, 2013). On the other hand, as the K72R mutant of E. coli exhibited (Britt et al, 2011), the N-terminal side of the central domain of RecA, which governs ATPase activity, is responsible for ATP hydrolysis.…”

“…The requirement for the ATP hydrolysis activity of RecA in IS256Bsu1 transposition may be similar to that of MuB in MuA transposition. According to the model of Mizuno et al (2013), the MuB filament on dsDNA is partially decomposed by MuB-ATP hydrolysis enhanced by MuA. The interaction of MuA and MuB nicks the terminus of the Mu genome and initiates transposition onto dsDNA exposed by MuA.…”