Gin-mediated site-specific recombination in bacteriophage Mu DNA: overproduction of the protein and inversion <i>in vitro</i>

Mertens, Gabriele; Hoffmann, Andrea; Blöcker, Helmut; Frank, Ronald; Kahmann, Regine

doi:10.1002/j.1460-2075.1984.tb02148.x

The EMBO Journal

1984

DOI: 10.1002/j.1460-2075.1984.tb02148.x

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Gin-mediated site-specific recombination in bacteriophage Mu DNA: overproduction of the protein and inversion in vitro

Gabriele Mertens

Andrea Hoffmann

Helmut Blöcker

et al.

Abstract: Inversion of the G segment in bacteriophage Mu DNA occurs by a site‐specific recombination event and determines the host specificity of Mu phage particles produced. Inversion is mediated by a Mu function (Gin). The gin gene has been placed under control of the inducible λ pL promoter and a synthetic Shine‐Dalgarno linker upstream of the initiation codon. The Gin protein content in induced cells is boosted to ˜10% of total protein. Partially purified extracts from overproducing strains promote efficient inversi… Show more

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Cited by 46 publications

(23 citation statements)

References 29 publications

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“…This is in keeping with the function of resolvase in the resolution of cointegrate structures (7,63). Conversely, the invertases are active in causing inversion of the DNA between two inverted target sites, whereas they cannot perform excision if the sites are in direct orientation (38)(39)(40)47). Again, this property is in keeping with the biological role of these proteins (40,64,66 Table 1 and Fig.…”

supporting

confidence: 59%

“…In this review I shall examine five such systems and point out similarities and differences among them. In addition to the A integrase system, these systems include the resolvases of transposons Tn3 and yb (60), the Cre protein of phage P1 (5), the Gin protein of phage Mu (47,58), the Hin protein of the phase inversion system of S. typhimurium (invertases) (38), and the FLP protein of the 2,um plasmid of S. cerevisiae (48,70).…”

mentioning

confidence: 99%

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Site-specific recombinases: changing partners and doing the twist

Sadowski¹

1986

J Bacteriol

195

109

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Thirty-five years have passed since the discovery of lysogenic induction of bacteriophage (46), and over the years it has become evident that site-specific integration of the phage chromosome into the bacterial genome is a key element of this process. While much of the genetic and biochemical data were derived from the phage lambda paradigm (73), site-specific recombination is not merely a curiosity confined to an arcane organism like lambda, but an important process controlling the development of many organisms as diverse as temperate bacteriophages and humans. The list of functions performed by site-specific recombination systems has grown to include, in addition to the integration and excision of bacteriophage chromosomes (15, 73), resolution of cointegrate structures generated during the "hopping" of transposons like Tn3 (7,60,61) (16,23,33).In hope of discovering universal biochemical mechanisms underlying these important processes, several groups have followed the lead of Nash and colleagues (53) and have developed systems that reproduce site-specific recombination in vitro. In this review I shall examine five such systems and point out similarities and differences among them. In addition to the A integrase system, these systems include the resolvases of transposons Tn3 and yb (60), the Cre protein of phage P1 (5), the Gin protein of phage Mu (47, 58), the Hin protein of the phase inversion system of S. typhimurium (invertases) (38), and the FLP protein of the 2,um plasmid of S. cerevisiae (48,70).Common features of site-specific recombinases. In his original formulation, Campbell (15) postulated that integration of the lambda genome occurred by reciprocal recombination between regions of homology on the circular phage chromosome and that of Escherichia coli. This region of exact homology is now known to be small, only 15 base pairs (bp), and crossing over occurs between the phage attachment site (attP [-240 bp long]) and its bacterial equivalent (attB [-25 bp long]) (see Fig. 1 and 2). The hallmark of all site-specific recombinases is that they promote reciprocal recombination (Fig. 1). In the lambda system, these sites cannot undergo further Intpromoted recombination without the help of the phage Xis protein whose function is necessary to perform the analogous function in vivo, namely excision of the prophage (2, 32, 41).An additional feature of the target sequences is their inherent asymmetry; they are not like the perfect palindromes recognized by many type II restriction endonucleases. This asymmetry imparts a directionality to the site-specific reactions. When two recombining sites are pointing in the same direction along the DNA molecule (direct orientation), recombination always results in excision of the DNA between the sites, whereas if the two sites point in opposite directions (inverse orientation), recombination inverts the DNA between the sites (Fig. 3).The recombination is catalyzed in vitro by the recombinase protein acting by itself (the resolvases, the Cre protein, and probably the FLP...

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supporting

confidence: 59%

mentioning

confidence: 99%

Site-specific recombinases: changing partners and doing the twist

Sadowski¹

1986

J Bacteriol

195

109

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“…RecA is a protein that promotes general homologous recombination by mediating homologous base pairing and single-strand DNA exchange. All of the previously recognized inversion systems are independent of RecA activity (8,22,26,34,41,43).…”

mentioning

confidence: 93%

Nested DNA inversion of Campylobacter fetus S-layer genes is recA dependent

Dworkin

Shedd

1997

J Bacteriol

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To explore this possibility, we cloned C. fetus recA and created mutant strains by marker rescue, in which recA is disrupted in either S ؉ or S ؊ strains. These mutants then were assessed for their abilities to alter SLP expression either in the presence or absence of a complementary shuttle plasmid harboring native recA. In contrast to all previously reported programmed DNA inversion systems, inversion in C. fetus is recA dependent.

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“…1) (10). The dependence of (Ϫ)supercoiling of substrate plasmids has also been well established in many related site-specific recombination systems such as Gin invertase (11,12), ␥␦ resolvase (13), Tn3 resolvase (14), and integrase (15).…”

mentioning

confidence: 96%

Hin-mediated Inversion on Positively Supercoiled DNA

Lim¹,

Lee²,

Jaxel³

et al. 1997

Journal of Biological Chemistry

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Hin recombinase requires negatively supercoiled DNA for an efficient inversion. We have generated positively supercoiled plasmid DNA using reverse gyrase from Sulfolobus shibatae and subjected it to the Hinmediated inversion reaction. Both Hin and Fis showed the same DNA binding activity regardless of the superhelical handedness of the substrate plasmid. However, inversion activity on positively supercoiled DNA was less than 1% of negatively supercoiled DNA. Assays designed to probe steps in inversion, showed that on positively supercoiled DNA, Hin was able to cleave the recombination sites with the same efficiency shown on negatively supercoiled DNA but was not able to exchange the cleaved DNA. Based on the theoretical differences between positive and negative supercoiling, our data may suggest that unwinding of the double helix at recombination sites is needed after DNA cleavage for strand exchange to occur.

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Gin-mediated site-specific recombination in bacteriophage Mu DNA: overproduction of the protein and inversion in vitro

Cited by 46 publications

References 29 publications

Site-specific recombinases: changing partners and doing the twist

Site-specific recombinases: changing partners and doing the twist

Nested DNA inversion of Campylobacter fetus S-layer genes is recA dependent

Hin-mediated Inversion on Positively Supercoiled DNA

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