Mismatch-targeted transposition of Mu: A new strategy to map genetic polymorphism

Yanagihara, Keiko; Mizuuchi, Kiyoshi

doi:10.1073/pnas.132403399

Cited by 48 publications

(46 citation statements)

References 13 publications

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“…7B)? Although MuA lacks sequence specificity, it is known to display a great preference for insertion in mismatch sites on the DNA (45). There is no physiological reason for such preference other than that the mismatch DNA would readily assume a deformed DNA structure, which is favored for the strand-transfer reaction.…”

Section: Discussionmentioning

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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Section: Discussionmentioning

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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“…One added benefit might be that higher supercoiling levels lead to rapid curing once E. coli is lysogenized by a prophage. Recent work shows that Mu transposition hotspots in Salmonella are different from those in E. coli (D. Manna, S. Porwollik, M. McClelland, and N. P. Higgins, submitted for publication), and lower negative supercoiling could account for different locations of unusual DNA structure that act as transposition hotspots (93). If the supercoiling set point is established by the gyrA and gyrB genes, it would be very interesting to see how Salmonella fitness and dichotomous growth rates would change after swapping the normal Salmonella gyrase alleles with E. coli gyrA and gyrB sequences.…”

Section: Discussionmentioning

confidence: 99%

Growth Rate Toxicity Phenotypes and Homeostatic Supercoil Control Differentiate Escherichia coli from Salmonella enterica Serovar Typhimurium

Champion

Higgins

2007

J Bacteriol

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Escherichia coli and Salmonella enterica serovar Typhimurium share high degrees of DNA and amino acid identity for 65% of the homologous genes shared by the two genomes. Yet, there are different phenotypes for null mutants in several genes that contribute to DNA condensation and nucleoid formation. The mutant R436-S form of the GyrB protein has a temperature-sensitive phenotype in Salmonella, showing disruption of supercoiling near the terminus and replicon failure at 42°C. But this mutation in E. coli is lethal at the permissive temperature. A unifying hypothesis for why the same mutation in highly conserved homologous genes of different species leads to different physiologies focuses on homeotic supercoil control. During rapid growth in mid-log phase, E. coli generates 15% more negative supercoils in pBR322 DNA than Salmonella. Differences in compaction and torsional strain on chromosomal DNA explain a complex set of single-gene phenotypes and provide insight into how supercoiling may modulate epigenetic effects on chromosome structure and function and on prophage behavior in vivo.Dichotomous growth is an adaptation that allows certain bacteria to divide rapidly under nutrient-rich conditions. During dichotomous growth, wild-type (WT) Escherichia coli can divide every 20 min, even though it takes Ͼ50 min to replicate the genome. Cells can divide rapidly only as long as initiation of bidirectional DNA synthesis with the DnaA "initiator" complex at the oriC "replicator" (42) is well coordinated with the formation of nucleoids and the distribution of replicated copies of the chromosome to each daughter cell at the time of partition. Recent studies with live cells show that a highly organized traffic pattern deposits each replisome arm at opposing edges of growing nucleoids (2, 90). At high growth rates, the average cell has a complex chromosome structure, with nucleoids containing two or four partially replicated arcs spanning the oriC initiator region. To distribute nucleoids, the cell division machinery needs to find the cell midpoint, decatenate all cross-links between chromosomal replicas at the dif site (19, 49), and move the nucleoids into each new daughter cell.The initiation frequency at oriC is critical for cell division. Hyperinitiation is toxic or lethal (77), with one problem being fork overrun. If a fork from a recent initiation cycle overtakes a previously initiated fork, large chromosome fragments with double-strand ends can be generated (4). We previously showed that the Salmonella gyrB652 mutation causes "growth rate toxicity" that includes topological chaos near the dif site (68). Gyrase hypomorphs in Salmonella lose supercoiling near the terminus of replication, even though plasmids in the same cell maintain near-normal superhelical densities (68). Recent experiments suggest that a similar fate may occur in gyrase mutants of E. coli (29,45).The GyrB protein is highly conserved in E. coli and Salmonella. Of its 804 amino acids, 97% are identical in the two species, with most substitutions being ...

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“…The final reaction conditions were 100 ng of genomic DNA, 200 M each dNTP, 1.0 mM MgCl 2 , 0.66 M each primer, and 0.4 U of TaqDNA polymerase (Invitrogen). The Mu transposition reactions were previously described (Yanagihara and Mizuuchi, 2002). The Mu transpososome mixture was prepared by mixing 300 nM MuA transposase, 100 nM labeled Mu-end DNA, and 25 mM HEPES, pH 7.6, 15% (v/v) glycerol, 15% DMSO, 10 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and 156 mM NaCl.…”

Section: Methodsmentioning

confidence: 99%

A Missense Mutation of the Gene Encoding Voltage-Dependent Sodium Channel (Na_v1.1) Confers Susceptibility to Febrile Seizures in Rats

Mashimo¹,

Ohmori²,

Ouchida³

et al. 2010

J. Neurosci.

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Although febrile seizures (FSs) are the most common convulsive syndrome in infants and childhood, the etiology of FSs has remained unclarified. Several missense mutations of the Na v 1.1 channel (SCN1A), which alter channel properties, have been reported in a familial syndrome of GEFSϩ (generalized epilepsy with febrile seizures plus). Here, we generated Scn1a-targeted rats carrying a missense mutation (N1417H) in the third pore region of the sodium channel by gene-driven ENU (N-ethyl-N-nitrosourea) mutagenesis. Despite their normal appearance under ordinary circumstances, Scn1a mutant rats exhibited remarkably high susceptibility to hyperthermiainduced seizures, which involve generalized clonic and/or tonic-clonic convulsions with paroxysmal epileptiform discharges. Whole-cell patch-clamp recordings from HEK cells expressing N1417H mutant channels and from hippocampal GABAergic interneurons of N1417H mutant rats revealed a significant shift of the inactivation curve in the hyperpolarizing direction. In addition, clamp recordings clearly showed the reduction in action potential amplitude in the hippocampal interneurons of these rats. These findings suggest that a missense mutation (N1417H) of the Na v 1.1 channel confers susceptibility to FS and the impaired biophysical properties of inhibitory GABAergic neurons underlie one of the mechanisms of FS.

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Mismatch-targeted transposition of Mu: A new strategy to map genetic polymorphism

Cited by 48 publications

References 13 publications

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

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

Growth Rate Toxicity Phenotypes and Homeostatic Supercoil Control Differentiate Escherichia coli from Salmonella enterica Serovar Typhimurium

A Missense Mutation of the Gene Encoding Voltage-Dependent Sodium Channel (Na_v1.1) Confers Susceptibility to Febrile Seizures in Rats

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Mismatch-targeted transposition of Mu: A new strategy to map genetic polymorphism

Cited by 48 publications

References 13 publications

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

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

Growth Rate Toxicity Phenotypes and Homeostatic Supercoil Control Differentiate Escherichia coli from Salmonella enterica Serovar Typhimurium

A Missense Mutation of the Gene Encoding Voltage-Dependent Sodium Channel (Nav1.1) Confers Susceptibility to Febrile Seizures in Rats

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A Missense Mutation of the Gene Encoding Voltage-Dependent Sodium Channel (Na_v1.1) Confers Susceptibility to Febrile Seizures in Rats