Congruence of in Vivo and in Vitro Insertion Patterns in Hot E. coli Gene Targets of Transposable Element Mu: Opposing Roles of MuB in Target Capture and Integration

Ge, Jun; Harshey, Rasika M.

doi:10.1016/j.jmb.2008.05.032

Cited by 13 publications

(24 citation statements)

References 34 publications

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“…8A) (34). Consistent with this model, Mu transposition was observed at the junction of A/T and non-A/T DNA in vitro (97), and in the vicinity rather than within MuB bound regions in vivo (88). Two hot spots for Mu insertion near the bgl locus were also observed to be clustered at the borders of an A/T rich segment (92).…”

Section: Target Site Selectionsupporting

confidence: 57%

“…Two hot spots for Mu insertion near the bgl locus were also observed to be clustered at the borders of an A/T rich segment (92). Interestingly, the majority of A/T rich regions were unavailable for MuB binding in vivo because they are occupied by nucleoid proteins such as Fis, which have a similar binding preference (88, 97). Other strongly DNA-bound proteins also occlude Mu insertions (94).…”

Section: Target Site Selectionmentioning

confidence: 99%

“…7). It is not clear why MuB would bind strongly within the Mu genome, which is not A/T rich, albeit A/T content was shown to be an unreliable predictor of MuB binding in vivo ; MuB binding is expected to be modulated by host proteins in vivo (88, 97). A model for how formation of an independent 'Mu domain' might nucleate polymerization of MuB on the genome, forming a barrier against self-integration, has been proposed (Fig.…”

Section: Transposition Immunitymentioning

confidence: 99%

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Transposable Phage Mu

Harshey

2014

Microbiol Spectr

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Transposable phage Mu has played a major role in elucidating the mechanism of movement of mobile DNA elements. The high efficiency of Mu transposition has facilitated a detailed biochemical dissection of the reaction mechanism, as well as of protein and DNA elements that regulate transpososome assembly and function. The deduced phosphotransfer mechanism involves in-line orientation of metal ion-activated hydroxyl groups for nucleophilic attack on reactive diester bonds, a mechanism that appears to be used by all transposable elements examined to date. A crystal structure of the Mu transpososome is available. Mu differs from all other transposable elements in encoding unique adaptations that promote its viral lifestyle. These adaptations include multiple DNA (enhancer, SGS) and protein (MuB, HU, IHF) elements that enable efficient Mu end synapsis, efficient target capture, low target specificity, immunity to transposition near or into itself, and efficient mechanisms for recruiting host repair and replication machineries to resolve transposition intermediates. MuB has multiple functions, including target capture and immunity. The SGS element promotes gyrase-mediated Mu end synapsis, and the enhancer, aided by HU and IHF, participates in directing a unique topological architecture of the Mu synapse. The function of these DNA and protein elements is important during both lysogenic and lytic phases. Enhancer properties have been exploited in the design of mini-Mu vectors for genetic engineering. Mu ends assembled into active transpososomes have been delivered directly into bacterial, yeast, and human genomes, where they integrate efficiently, and may prove useful for gene therapy.

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Section: Target Site Selectionsupporting

confidence: 57%

Section: Target Site Selectionmentioning

confidence: 99%

Section: Transposition Immunitymentioning

confidence: 99%

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Transposable Phage Mu

Harshey

2014

Microbiol Spectr

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“…On natural DNA, which can range from 40% to 70% A/T, analysis of MuB binding and Mu insertion patterns is consistent with a fairly interspersed pattern of MuB binding, with insertions being directed to adjacent DNA sites free of MuB [19]. The pattern of Mu insertion within such DNA is unperturbed over a wide range of MuB concentrations.…”

Section: Discussionmentioning

confidence: 87%

Immunity of replicating Mu to self-integration: a novel mechanism employing MuB protein

Lou

Harshey

2010

Mobile DNA

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We describe a new immunity mechanism that protects actively replicating/transposing Mu from self-integration. We show that this mechanism is distinct from the established cis-immunity mechanism, which operates by removal of MuB protein from DNA adjacent to Mu ends. MuB normally promotes integration into DNA to which it is bound, hence its removal prevents use of this DNA as target. Contrary to what might be expected from a cis-immunity mechanism, strong binding of MuB was observed throughout the Mu genome. We also show that the cis-immunity mechanism is apparently functional outside Mu ends, but that the level of protection offered by this mechanism is insufficient to explain the protection seen inside Mu. Thus, both strong binding of MuB inside and poor immunity outside Mu testify to a mechanism of immunity distinct from cis-immunity, which we call 'Mu genome immunity'. MuB has the potential to coat the Mu genome and prevent auto-integration as previously observed in vitro on synthetic A/T-only DNA, where strong MuB binding occluded the entire bound region from Mu insertions. The existence of two rival immunity mechanisms within and outside the Mu genome, both employing MuB, suggests that the replicating Mu genome must be segregated into an independent chromosomal domain. We propose a model for how formation of a 'Mu domain' may be aided by specific Mu sequences and nucleoid-associated proteins, promoting polymerization of MuB on the genome to form a barrier against self-integration.

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“…In addition to facilitating protein polymerization through intra-filament interactions, the ability of the NTD in bridging MuB filaments expands the possibilities of the dynamic association of MuB with the DNA. It has been found that the DNA inside the filament cannot be used as a target DNA for transposition (Ge and Harshey, 2008;Mizuno et al, 2013) and a mechanism was proposed by which the coordinated ATP hydrolysis of several MuB subunits at the filament end could distort the DNA structure, favoring DNA bending and recognition by the transposome (Mizuno et al, 2013). The interaction between MuB filaments bound to nearby DNA sequences could further facilitate the bending of the intermediate sequence or the formation of a special DNA structure, thus assisting target DNA capture by MuA.…”

Section: Discussionmentioning

confidence: 99%