A truncated form of the bacteriophage Mu B protein promotes conservative integration, but not replicative Transposition, of Mu DNA

Chaconas, George; Giddens, Elizabeth B.; Miller, Janet L.; Gloor, Gregory B.

doi:10.1016/s0092-8674(85)80066-0

Cited by 46 publications

(30 citation statements)

References 30 publications

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“…In addition, a Mu B mutant lacking residues 295-312 from the C-terminal region of the protein is also in this functional class (50). Interestingly, this protein is able to promote integrative but not replicative transposition in vivo (39,50).…”

Section: Discussionmentioning

confidence: 99%

“…Mutations in the N-terminal domain of Mu B have been shown to affect target capture, but these mutants retain their ability to interact with the transpososome complex (37,38). A small truncation of the C-terminal domain of Mu B blocks replicative transposition but not integration, whereas longer truncations affect both replicative and integrative transposition in vivo (39).…”

mentioning

confidence: 99%

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Effect of Mutations in the C-terminal Domain of Mu B on DNA Binding and Interactions with Mu A Transposase

Coros

Sekino

Baker

et al. 2003

Journal of Biological Chemistry

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Effect of Mutations in the C-terminal Domain of Mu B on DNA Binding and Interactions with Mu A Transposase

Coros

Sekino

Baker

et al. 2003

Journal of Biological Chemistry

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“…MuA then assembles into a series of nucleoprotein complexes, collectively referred to as transpososomes, that are responsible for catalyzing the DNA processing reactions required to complete transposition (4 -7). A second protein, the ATPase MuB, is also required to complete the transposition reaction (8,9).…”

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confidence: 99%

Visualizing the Assembly and Disassembly Mechanisms of the MuB Transposition Targeting Complex

Greene

Mizuuchi

2004

Journal of Biological Chemistry

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MuB, a protein essential for replicative DNA transposition by the bacteriophage Mu, is an ATPase that assembles into a polymeric complex on DNA. We used total internal reflection fluorescence microscopy to observe the behavior of MuB polymers on single molecules of DNA. We demonstrate that polymer assembly is initiated by a stochastic nucleation event. After nucleation, polymer assembly occurs by a mechanism involving the sequential binding of small units of MuB. MuB that bound to A/T-rich regions of the DNA assembled into large polymeric complexes. In contrast, MuB that bound outside of the A/T-rich regions failed to assemble into large oligomeric complexes. Our data also show that MuB does not catalyze multiple rounds of ATP hydrolysis while remaining bound to DNA. Rather, a single ATP is hydrolyzed, then MuB dissociates from the DNA. Finally, we show that "capping" of the enhanced green fluorescent protein-MuB polymer ends with unlabeled MuB dramatically slows, but does not halt, dissociation. This suggests that MuB dissociation occurs through both an end-dependent mechanism and a slower mechanism wherein subunits dissociate from the polymer interior.DNA transposition is used by many organisms to regulate the dispersion of their genetic material throughout a host cell population (1, 2). The bacteriophage Mu transposition reaction is initiated by the binding of the transposase protein MuA to specific sites at the ends of the phage genome (3). MuA then assembles into a series of nucleoprotein complexes, collectively referred to as transpososomes, that are responsible for catalyzing the DNA processing reactions required to complete transposition (4 -7). A second protein, the ATPase MuB, is also required to complete the transposition reaction (8, 9).MuB performs multiple functions during transposition, including stimulating assembly of the transpososomes, stimulating the MuA catalyzed DNA processing reactions, influencing the selection of the DNA target site, and regulating the disassembly of the transpososome (10 -13). MuB does not share extensive sequence similarity with any other known protein.However, like many other ATPases, MuB does contain two conserved amino acid motifs, Walker A and B boxes, that are essential for the binding and hydrolysis of nucleotide triphosphates (14, 15). In the presence of ATP, MuB assembles into large oligomeric complexes on DNA, making the bound DNA an efficient target for strand transfer (16,17). MuB accumulates on DNA that is not bound by MuA, resulting in a strong preference for transposition to occur at DNA sites at least 10 -20 kb from MuA-bound regions (12,18). This phenomenon is called target immunity and provides a critical mechanism for Mu to prevent disruption of its genome through an aberrant transposition event.We have established a system that allows us to observe single complexes of MuB bound to an immobilized molecule of DNA (17). We have also demonstrated that MuB forms a large oligomer on DNA and that these oligomers were tightly bound to A/T-rich sequences (17)...

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“…The A protein eluted around 530 mM KC1. The B protein of bacteriophage Mu was purified as described (16). For overproduction of the A and B proteins either strain NF1 with plasmid pGP603 or strain SG13508 with plasmid MK209 was used.…”

Section: Protein Purification and In Vitro Transpositionmentioning

confidence: 99%

“…Effect of the B protein on the interaction of the transposase with the left end of Mu Chaconas et al (16) showed that the B protein of Mu has no preference for Mu DNA and that it binds to both ssDNA and dsDNA with the same affinity. Since the B protein is also essential in an early step of transposition (14), we also examined the simultaneous interaction of the A and B proteins with the left end of Mu.…”

Section: Dnasei Footprinting Experimentsmentioning

confidence: 99%

Interactions of the transposase with the ends of Mu: formation of specific nucleoprotein structures and non-cooperative binding of the transposase to its binding sites

et al. 1987

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Transposition of the E.coli bacteriophage Mu requires the phage encoded A and B proteins, the host protein HU and the host replication proteins. The ends of the genome of the phage, on which some of these proteins act, both contain three transposase (A) binding sites. The organization of these binding sites on each end, however, is different. Here we show, using DNase footprinting experiments with purified A protein, that mutant A binding sites, which affect transposition, have decreased affinity for the transposase. Furthermore the transposase binds non-cooperatively to all A binding sites both in the left and right end of Mu. Electron microscopic studies show that the A protein forms specific nucleoprotein structures upon binding to the ends of Mu. The A and B proteins interact with the ends of Mu to generate larger structures than with the A protein alone.

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A truncated form of the bacteriophage Mu B protein promotes conservative integration, but not replicative Transposition, of Mu DNA

Cited by 46 publications

References 30 publications

Effect of Mutations in the C-terminal Domain of Mu B on DNA Binding and Interactions with Mu A Transposase

Effect of Mutations in the C-terminal Domain of Mu B on DNA Binding and Interactions with Mu A Transposase

Visualizing the Assembly and Disassembly Mechanisms of the MuB Transposition Targeting Complex

Interactions of the transposase with the ends of Mu: formation of specific nucleoprotein structures and non-cooperative binding of the transposase to its binding sites

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