The Mu Enhancer Is Functionally Asymmetric Both incis and in trans

Jiang, Haowei; Harshey, Rasika M.

doi:10.1074/jbc.m008523200

Cited by 11 publications

(10 citation statements)

References 46 publications

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“…The strong bias towards one particular configuration of sites constitutes a topological filter. Topological filters have been observed in several other recombination systems such as the Tn3/ϒδ resolvases, the hin/gin/cin inversion systems, and phage Mu transposition (59,60,61,62,63). These are similarly dependent on negative supercoiling in the substrate.…”

Section: Dna Topologymentioning

confidence: 85%

Mariner and the ITm Superfamily of Transposons

2015

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The IS630-Tc1-mariner (ITm) family of transposons is one of the most widespread in nature. The phylogenetic distribution of its members shows that they do not persist for long in a given lineage, but rely on frequent horizontal transfer to new hosts. Although they are primarily selfish genomic-parasites, ITm transposons contribute to the evolution of their hosts because they generate variation and contribute protein domains and regulatory regions. Here we review the molecular mechanism of ITm transposition and its regulation. We focus mostly on the mariner elements, which are understood in the greatest detail owing to in vitro reconstitution and structural analysis. Nevertheless, the most important characteristics are probably shared across the grouping. Members of the ITm family are mobilized by a cut-and-paste mechanism and integrate at 5′-TA dinucleotide target sites. The elements encode a single transposase protein with an N-terminal DNA-binding domain and a C-terminal catalytic domain. The phosphoryl-transferase reactions during the DNA-strand breaking and joining reactions are performed by the two metal-ion mechanism. The metal ions are coordinated by three or four acidic amino acid residues located within an RNase H-like structural fold. Although all of the strand breaking and joining events at a given transposon end are performed by a single molecule of transposase, the reaction is coordinated by close communication between transpososome components. During transpososome assembly, transposase dimers compete for free transposon ends. This helps to protect the host by dampening an otherwise exponential increase in the rate of transposition as the copy number increases.

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Section: Dna Topologymentioning

confidence: 85%

Mariner and the ITm Superfamily of Transposons

2015

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“…28 Previous investigations employed two strategies to probe the interactions of MuA monomers bound to specific sub-sites at the left and right ends with the O1 and O2 operator regions of the enhancer. 5,6,24,29 One employed substitution/deletion analyses of individual sites; the other utilized two transposases that share a common end-binding specificity but have distinct enhancer-binding specificities together with bi-specific hybrid enhancers. The outcomes from these studies, along with the finding that a MuA variant lacking the enhancer-binding domain can be incorporated into a functional transpososome, 15 imply that only a subset of the MuA monomers associated with the Mu ends needs to interact with the enhancer.…”

Section: Discussionmentioning

confidence: 99%

“…These products (type II) have been characterized extensively in a number of earlier studies, and are recognized easily by their relative mobility with respect to type I. 6,25,26 The yield of the type I complex, and by inference that of the type 0 complex from which it is quantitatively derived, in a 30 min reaction was roughly 70-90% of the input substrate. However, as a precaution against potential alterations of DNA topology within the transpososome due to DNA cleavage, the assays displayed in Figure 5(b)-(d) and Figure 6 were carried out by replacing Mg 2+ with Ca 2+ to block the conversion of type 0 into type I.…”

Section: Experimental Logic For Mapping the Interactions Between The mentioning

confidence: 98%

“…4 An essential interaction between R1 and O1 inferred previously by use of hybrid transposases and hybrid enhancers is consistent with this assembly scheme. 5,6 The L end is then captured by the ER complex in the presence of HU, and a DNA crossing is introduced between E and L. Two more DNA crossings are trapped by the interwrapping of L and R to complete the LER synapse, in which a total of five supercoil nodes are sequestered. 7 Footprinting experiments suggest that L1 is the last sub-site to join this complex, delivered by HUpromoted DNA bending at the L end.…”

Section: Introductionmentioning

confidence: 99%

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Interactions of Phage Mu Enhancer and Termini that Specify the Assembly of a Topologically Unique Interwrapped Transpososome

Yin¹,

Suzuki²,

Lou³

et al. 2007

Journal of Molecular Biology

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“…For the Mu transposase (MuA), two different enhancer-independent situations have been described. One involves an enhancer-independent transposase that, unlike the invertase and resolvase systems, does not relieve the dependence on DNA supercoiling or on the correct orientation of Mu ends (15,16). The other involves addition of Me 2 SO (dimethyl sulfoxide) to the reaction; this situation does provide independence from topological and directional restrictions (17).…”

mentioning

confidence: 99%

Enhancer-independent Mu transposition from two topologically distinct synapses

Yin

Harshey

2005

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

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Transposition of Mu is strictly dependent on a specific orientation of the left (L) and right (R) ends of Mu and a distant enhancer site (E) located on supercoiled DNA. Five DNA crossings are trapped in the three-site synapse, two of which are contributed by the interwrapping of L and R. To determine the contribution of E to the interwrapping of Mu ends, we examined the topology of the LR synapse under two different enhancer-independent reaction conditions. One of these conditions, which also alleviates the requirement for a specific orientation of Mu ends, revealed two topologically distinct arrangements of the ends. In their normal relative orientation, L and R were either plectonemically interwrapped or aligned by random collision. Addition of the enhancer to this system channeled synapsis toward the interwrapped pathway. When the ends were in the wrong relative orientation, synapsis occurred exclusively by random collision. In the second enhancerindependent condition, which retains the requirement for a specific orientation of Mu ends, synapsis of L and R was entirely by interwrapping. The two distinct kinds of synapses also were identified by gel electrophoresis. We discuss these results in the context of the ''topological filter'' model and consider the many contributions the enhancer makes to the biologically relevant interwrapped synapse.DNA topology ͉ topological filter ͉ Mu transposase ͉ Cre recombination S ite-specific recombination systems have provided a microscope for understanding how distant DNA segments interact with each other. Some of these systems are highly selective with respect to orientation of the interacting sites and recombine through a very specific synapse topology. Examples include phage Mu transposase and several resolvase and invertase systems (1-8). How is their specific topology selected? What distinguishes these systems from more permissive ones such as Flp recombinase and phage integrase, which can interact by random collision and recombine through a spectrum of DNA topologies (9, 10)? A hallmark of the former systems is the participation of three sites in the productive synapse, whereas the latter systems involve interaction of two sites. A three-site synapse on a circular, supercoiled substrate is thought to arise by DNA slithering or branching rather than by random collision and can be highly restrictive with respect to the permissible orientation of the interacting sites.There exist mutant invertases and resolvases that do not require complex synapse architecture or substrate circularity and give random-collision recombination products. These proteins have acquired independence from the enhancer or accessory binding sites (11)(12)(13)(14). For the Mu transposase (MuA), two different enhancer-independent situations have been described. One involves an enhancer-independent transposase that, unlike the invertase and resolvase systems, does not relieve the dependence on DNA supercoiling or on the correct orientation of Mu ends (15,16). The other involves addition of Me 2 SO (d...

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The Mu Enhancer Is Functionally Asymmetric Both incis and in trans

Cited by 11 publications

References 46 publications

Mariner and the ITm Superfamily of Transposons

Mariner and the ITm Superfamily of Transposons

Interactions of Phage Mu Enhancer and Termini that Specify the Assembly of a Topologically Unique Interwrapped Transpososome

Enhancer-independent Mu transposition from two topologically distinct synapses

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