DNA-promoted assembly of the active tetramer of the Mu transposase.

Baker, Tania A.; Mizuuchi, Kiyoshi

doi:10.1101/gad.6.11.2221

Cited by 110 publications

(84 citation statements)

References 30 publications

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“…Gel filtration and sucrose sedimentation revealed that the purified transposase forms a dimer in a solution containing 0.5 M NaCl. We assume that Tn3 transposase binds in a dimeric form to each of two IR sequences of Tn3 and mediates to give rise to a synaptic complex or a transpososome, which consists of two IRs and the transposase in a tetrameric form, such as MuA transposase in a transposition reaction (Baker & Mizuuchi 1992).…”

Section: Discussionmentioning

confidence: 99%

**Specific nicking at the 3′ ends of the terminal inverted repeat sequences in transposon Tn3 by transposase and an E. coli protein ACP**

1996

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Background: Tn3, a bacterial transposon, carries tnpA gene encoding transposase which is essential for its transposition. The transposition of Tn3 has been reproduced in vitro in a cell extract containing transposase by using a plasmid carrying mini-Tn3 as the donor and another plasmid as the target. Transposase has the ability to bind to the 38-bp terminal inverted repeats (IRs) of Tn3. The molecular mechanism of the initiation step of the Tn3 transposition reaction promoted by the transposase has, however, not been understood.

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Section: Discussionmentioning

confidence: 99%

**Specific nicking at the 3′ ends of the terminal inverted repeat sequences in transposon Tn3 by transposase and an E. coli protein ACP**

1996

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“…HU is known to stimulate assembly of prepriming replication complexes in vitro IBaker et al 1986;Skarstad et al 1990), as well as the assembly of recombination intermediates in phage Mu transposition (Lavoie and Chaconas 1990;Baker and Mizuuchi 1992) and Hin-mediated inversion (Johnson et al 1986;Haykinson and Johnson 1993). The known properties of HU, HMG1, and HMG2 are distinct from their sequence-specific counterparts: IHF in bacteria and LEF1 (the clearest example) in mammals.…”

Section: Discussionmentioning

confidence: 99%

The nonspecific DNA-binding and -bending proteins HMG1 and HMG2 promote the assembly of complex nucleoprotein structures.

1993

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The mammalian high mobility group proteins HMG1 and HMG2 are abundant, chromatin-associated proteins whose cellular function is not known. In this study we show that these proteins can substitute for the prokaryotic DNA-bending protein HU in promoting the assembly of the Hin invertasome, an intermediate structure in Hin-mediated site-specific DNA inversion. Formation of this complex requires the assembly of the Hin recombinase, the Fis protein, and three cis-acting DNA sites, necessitating the looping of intervening DNA segments. Invertasome assembly is strongly stimulated by HU or HMG proteins when one of these segments is shorter than 104 bp. By use of ligase-mediated circularization assays, we demonstrate that HMG1 and HMG2 can bend DNA extremely efficiently, forming circles as small as 66 bp, and even 59-bp circles at high HMG protein concentrations. In both invertasome assembly and circularization assays, substrates active in the presence of HMG1 contain one less helical turn of DNA compared with substrates active in the presence of HU protein. Analysis of different domains of HMG1 generated by partial proteolytic digestion indicate that DNA-binding domain B is sufficient for both bending and invertasome assembly. We suggest that an important biological function of HMG1 and HMG2 is to facilitate cooperative interactions between cis-acting proteins by promoting DNA flexibility. A general role for HMG1 and HMG2 in chromatin structure is also suggested by their ability to wrap DNA duplexes into highly compact forms.

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“…But, MuA recognizes the recombination sites at the ends of the transposable element with a different domain of the protein, and then assembles a tetramer that is bound to both ends of the element on a supercoiled DNA substrate (25,26). The interactions of MuA with the end recombination sites actually promote tetramer formation, and it is this complex that is active for recombination (27). In fact, under altered reaction conditions MuA can form a tetramer upon binding a single recombination site, but not on the enhancer sites (27).…”

Section: Discussionmentioning

confidence: 99%

“…The interactions of MuA with the end recombination sites actually promote tetramer formation, and it is this complex that is active for recombination (27). In fact, under altered reaction conditions MuA can form a tetramer upon binding a single recombination site, but not on the enhancer sites (27). Therefore, when we consider the nature of the MBP-Piv complexes with sub and invL, it is possible that the sub site is an assembly site for the multimeric form of Piv that binds to the recombination sites.…”

Section: Discussionmentioning

confidence: 99%

Multiple DNA Binding Activities of the Novel Site-specific Recombinase, Piv, from Moraxella lacunata

Tobiason

Lenich

Glasgow

1999

Journal of Biological Chemistry

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The recombinase, Piv, is essential for site-specific DNA inversion of the type IV pilin DNA segment in Moraxella lacunata and Moraxella bovis. Piv shows significant homology with the transposases of the IS110/ IS492 family of insertion elements, but, surprisingly, Piv contains none of the conserved amino acid motifs of the Int or Hin/Res families of site-specific recombinases. Therefore, Piv may mediate site-specific recombination by a novel mechanism. To begin to determine how Piv may assemble a synaptic nucleoprotein structure for DNA cleavage and strand exchange, we have characterized the interaction of Piv with the DNA inversion region of M. lacunata. Gel shift and nuclease/chemical protection assays, competition and dissociation rate analyses, and cooperativity studies indicate that Piv binds two distinct recognition sequences. One recognition sequence, found at multiple sites within and outside of the invertible segment, is bound by Piv protomers with high affinity. The second recognition sequence is located at the recombination cross-over sites at the ends of the invertible element; Piv interacts with this sequence as an oligomer with apparent low affinity. A model is proposed for the role of the different Piv binding sites of the M. lacunata inversion region in the formation of an active synaptosome.Numerous site-specific DNA recombination systems and DNA transposition systems have been characterized biochemically and have been found to follow two distinct chemical pathways for DNA cleavage and strand transfer in recombination (reviewed in Refs. 1-4). Site-specific recombination, mediated by the recombinases of the -integrase and Hin/resolvase families, involves a two-step transesterification reaction in which the intermediate is a covalent recombinase-DNA linkage. This covalent attachment is the result of nucleophilic attack on the DNA phosphodiester backbone by a hydroxyl group of the conserved serine (Hin/resolvase), or tyrosine (-integrase), of the recombinase. In the second transesterification reaction, the phosphodiester linkages of the exchanged DNA strands are restored (reviewed in Refs. 2 and 3). In contrast, DNA transposition, mediated by transposases containing the catalytic DDE amino acid motif, utilizes a hydrolysis reaction for cleavage at the ends of the transposable element. This first cleavage leaves 3Ј-OH ends to act directly as the attacking nucleophile in a one-step trans-esterification reaction resulting in strand exchange. Resolution of the transposition process involves DNA replication or DNA repair activity to fill in gaps left at the target site due to the staggered cut mediated by the transposase and the 3Ј hydroxyl groups at the element ends (reviewed in Refs. 1 and 4).These features of the recombination reactions mediated by site-specific recombinases and transposases suggest that a group of related recombinases would not mediate both sitespecific recombination and transposition. Therefore, it is surprising that the site-specific recombinase Piv, which directs site-specific DN...

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DNA-promoted assembly of the active tetramer of the Mu transposase.

Cited by 110 publications

References 30 publications

**Specific nicking at the 3′ ends of the terminal inverted repeat sequences in transposon Tn3 by transposase and an E. coli protein ACP**

**Specific nicking at the 3′ ends of the terminal inverted repeat sequences in transposon Tn3 by transposase and an E. coli protein ACP**

The nonspecific DNA-binding and -bending proteins HMG1 and HMG2 promote the assembly of complex nucleoprotein structures.

Multiple DNA Binding Activities of the Novel Site-specific Recombinase, Piv, from Moraxella lacunata

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