Characterization of amber mutations in bacteriophage Mu transposase: a functional analysis of the protein

Desmet, Lucie; Faelen, Michel; Gama, M.-J.; Ferhat, A; Toussaint, Ariane

doi:10.1111/j.1365-2958.1989.tb00265.x

Cited by 17 publications

(7 citation statements)

References 42 publications

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“…These data suggest that the amino acid sequence at the extreme carboxyl terminus of MuA is important for disassembly and may be recognized directly by ClpX. The carboxy-terminal domain of MuA is required for replicative transposition in vivo, but is not essential for the nonreplicative transposition that occurs during infection (Betermier et al 1989;Desmet et al 1989) The last 8 amino acids at the carboxyl terminus of MuA are QNRRKKAI (Harshey et al 1995). This sequence appeared similar to the carboxy-terminal amino acidic sequence of XO protein (Sanger et al 1982), PI Phd protein (Lehnherr et al 1993;Lehnherr and Yarmolinsky 1995), and the virulent derivatives of the Mu repressor (Geuskens et al 1992;Laachouch et al 1995); these proteins are all known or suspected substrates of the ClpXP protease.…”

Section: The Carboxyl Terminus Of Mua Is Required For Disassembly By mentioning

confidence: 99%

Disassembly of the Mu transposase tetramer by the ClpX chaperone.

Levchenko¹,

Luo²,

Baker³

1995

Genes Dev.

266

241

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Mu transposition is promoted by an extremely stable complex containing a tetramer of the transposase (MuA) bound to the recombining DNA. Here we purify the Escherichia coli ClpX protein, a member of a family of multimeric ATPases present in prokaryotes and eukaryotes (the Clp family), on the basis of its ability to remove the transposase from the DNA after recombination. Previously, ClpX has been shown to function with the ClpP peptidase in protein turnover. However, neither ClpP nor any other protease is required for disassembly of the transposase. The released MuA is not modified extensively, degraded, or irreversibly denatured, and is able to perform another round of recombination in vitro. We conclude that ClpX catalyzes the ATP-dependent release of MuA by promoting a transient conformational change in the protein and, therefore, can be considered a molecular chaperone. ClpX is important at the transition between the recombination and DNA replication steps of transposition in vitro; this function probably corresponds to the essential contribution of ClpX for Mu growth. Deletion analysis reveals that the sequence at the carboxyl terminus of MuA is important for disassembly by ClpX and can target MuA for degradation by ClpXP in vitro. These data contribute to the emerging picture that members of the Clp family are chaperones specifically suited for disaggregating proteins and are able to function with or without a collaborating protease.

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Section: The Carboxyl Terminus Of Mua Is Required For Disassembly By mentioning

confidence: 99%

Disassembly of the Mu transposase tetramer by the ClpX chaperone.

Levchenko¹,

Luo²,

Baker³

1995

Genes Dev.

266

241

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“…Eleven positions within MuA were modified. Residues were selected for mutagenesis as follows: (i) previous deletion analysis showing the importance of the amino acids between residues 574 and 605 (18,36), (ii) the position of a mutation that renders MuA defective in strand transfer at residue 548 (37), and (iii) possible amino acid sequence similarities with other transposition proteins (Nancy Craig and Lars Sundstr6m, personal communication; see Fig. 4).…”

mentioning

confidence: 99%

Identification of residues in the Mu transposase essential for catalysis.

Baker

Luo²

1994

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

125

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“…The 10 kDa C-terminal domain has been implicated in Mu A-Mu B interactions by genetic studies (Harshey and Cuneo, 1987;Toussaint et al, 1987;Btermier et al, 1987Btermier et al, , 1989. The 35 kDa core possesses non-specific DNA binding activity (Nakayama et al, 1987) and is important in the stabilization of specific A binding at the ends of Mu DNA in vitro (B6termier et al, 1989) and presumably in vivo (Desmet et al, 1989). The Mu A N-terminal domain is -30 kDa and confers sequence specific DNA binding properties (Nakayama et al, 1987) at the Mu ends as well as at the enhancer-like element via two different protein subdomains (Leung et al, 1989;Mizuuchi and Mizuuchi, 1989).…”

mentioning

confidence: 99%

Structural aspects of a higher order nucleoprotein complex: induction of an altered DNA structure at the Mu-host junction of the Mu type 1 transpososome.

et al. 1991

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The Mu in vitro strand transfer reaction proceeds via two stable higher order nucleoprotein complexes, the Type 1 and Type 2 transpososomes. The Mu A protein is responsible for the structural and functional integrity of the Type 1 transpososome. We have investigated the quaternary structure of the Mu A protein within this complex by chemical cross‐linking experiments and found that the basic structural unit is an A tetramer. Three Mu A binding sites in the transpososome are protected by DNase I footprinting: the outermost A binding sites L1 and R1, as well as R2. Genetic evidence is also presented which corroborates this result. Efficient formation of Type 1 complexes occurs in mini‐Mus with the L3 or R3 sites deleted or when the L2 site has been substituted; but no reaction occurs in the absence of R2. The protection at the L1 and R1 sites extends 12–13 bp beyond the Mu‐host junctions as seen by DNase I and methidiumpropyl‐EDTA.Fe(II) [MPE.Fe(II)] foot‐printing, indicating Mu A contacts with the flanking host sequences in the transpososome but not on linear DNA; furthermore, hydroxyl radical footprinting shows an unprecedentedly large enhancement on the continuous strand, 2 bp beyond the nick site outside the Mu right end, which suggests that an altered DNA structure is induced upon Type 1 complex formation.

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Characterization of amber mutations in bacteriophage Mu transposase: a functional analysis of the protein

Cited by 17 publications

References 42 publications

Disassembly of the Mu transposase tetramer by the ClpX chaperone.

Disassembly of the Mu transposase tetramer by the ClpX chaperone.

Identification of residues in the Mu transposase essential for catalysis.

Structural aspects of a higher order nucleoprotein complex: induction of an altered DNA structure at the Mu-host junction of the Mu type 1 transpososome.

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