Properties of a mutant Cre protein that alters the topological linkage of recombination products

Abremski, Kenneth; Frommer, Beth R.; Wierzbicki, Anna; Hoess, Ronald H.

doi:10.1016/0022-2836(88)90518-9

Cited by 27 publications

(25 citation statements)

References 14 publications

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“…In the Cre lox system (8) and in the bacteriophage X integrase system (9, 10), there is genetic evidence that site-specific recombination stimulates gene conversion of nearby markers. In both these systems, studies of linking number changes in DNA during recombination (11, 12) and the ability of the recombinases to act as type I topoisomerases (13,14) are consistent with a model of sequential single-stranded breaks and exchanges. In addition, artificial Holliday intermediates have been formed using the DNA ofthe bacteriophage X att site, and it has been demonstrated that such structures are acted upon by the recombinase Int (15).…”

Section: Introductionmentioning

confidence: 69%

Isolation and characterization of intermediates in site-specific recombination.

Hoess

Wierzbicki

Abremski

1987

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

135

104

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Cre, the site-specific recombinase from bacteriophage P1, catalyzes a recombination reaction between specific DNA sequences designated as lox sites. The breakage and rejoining of partners during this recombination process must be highly concerted because it has not been possible to detect intermediates ofthe reaction with wild-type Cre. Several mutant Cre proteins have been isolated that produce significant amounts of a possible intermediate product of the recombination reaction. The product has been identified as a Holliday structure in which one set of the DNA strands of the recombining partners has been exchanged. Wild-type Cre protein is capable of acting on this structure to form recombinant products, which is consistent with this being an intermediate in the recombination reaction. Characterization of the Holliday structure indicated that one set of strands in the recombiming partners was always exchanged preferentially before the other set. In addition, it has been found that certain Cre mutants that are unable to carry out recombination in vitro are able to resolve the intermediate. This suggests that these mutants are defective in a step in the reaction that precedes the formation of the Holliday intermediate.Genetic recombination requires the physical exchange of DNA strands among partners in the reaction. The development of well-defined in vitro recombination systems has offered the possibility of biochemically dissecting this complex reaction (reviewed in refs. 1 and 2). Of considerable importance in understanding the mechanism of recombination is the identification and isolation of intermediates in the reaction. Because of its relative simplicity, we have chosen the Cre lox site-specific recombination system of bacteriophage P1 as a model to study this reaction. The system consists of a single protein, Cre, which mediates the recombination process between two 34-base-pair (bp) sequences designated as lox sites (3). Each lox site is comprised of two 13-bp inverted repeats separated by an 8-bp spacer region (4). Upon binding to the inverted repeats, Cre cleaves the DNA in the spacer region to initiate strand exchange with an equivalent partner in the reaction (5).Early concepts of genetic recombination involved a simple breakage and rejoining mechanism, in which double-stranded breaks were formed and all partners in the reaction were exchanged at once. The discovery of gene conversion in fungi, which could not be adequately explained by simple breakage and rejoining, stimulated the formulation of an alternative model (6). This model proposed that recombination took place by sequential exchange, with first one set of strands being cleaved and exchanged followed by similar reactions with the second set of strands. The model predicts that between those two events a cross-stranded structure or Holliday intermediate will be formed (6). A large body ofboth genetic and physical evidence has implicated the Holliday structure as an intermediate in recombination. Perhaps most convincing are the direct o...

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Section: Introductionmentioning

confidence: 69%

Isolation and characterization of intermediates in site-specific recombination.

Hoess

Wierzbicki

Abremski

1987

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

135

104

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“…Cre can mediate recombination between cryptic loxP sites naturally present in E. coli, yeast, and mammalian genomes (23)(24)(25)(26), and earlier studies already reported the induction of nicks and breaks after incubation of cryptic loxP sites with purified Cre recombinase in vitro (15). Given that mammalian genomes contain multiple cryptic loxP sites (26), the inadvertent effects of Cre presented here may be explained by aberrant Cre activity on these sites.…”

Section: Discussionmentioning

confidence: 90%

“…Mutation analysis has revealed that for efficient recombination between loxP sites, homology of the spacer region is required (12). Recombination between a mutant loxP site, containing a deletion in the spacer region, and a wild-type loxP site in vitro with purified Cre leads to the formation of single-and doublestrand breaks (15).…”

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confidence: 99%

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Growth inhibition and DNA damage induced by Cre recombinase in mammalian cells

Loonstra

Vooijs²,

Beverloo³

et al. 2001

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

539

473

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The use of Cre͞loxP recombination in mammalian cells has expanded rapidly. We describe here that Cre expression in cultured mammalian cells may result in a markedly reduced proliferation and that this effect is dependent on the endonuclease activity of Cre. Chromosome analysis after Cre expression revealed numerous chromosomal aberrations and an increased number of sister chromatid exchanges. Titration experiments in mouse embryo fibroblasts with a ligand-regulatable Cre-ER T show that toxicity is dependent on the level of Cre activity. Prolonged, low levels of Cre activity permit recombination without concomitant toxicity. This urges for a careful titration of Cre activity in conditional gene modification in mammalian cells.genotoxicity ͉ conditional knockout

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“…72 When Cre111 recombines a supercoiled substrate, the knotted and catenated products are, in their conditions, significantly more complex than those produced by wild-type Cre.…”

Section: 2mentioning

confidence: 99%

Predicting Knot or Catenane Type of Site-Specific Recombination Products

Buck¹,

Flapan²

2007

Journal of Molecular Biology

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Site-specific recombination on supercoiled circular DNA yields a variety of knotted or catenated products. We develop a model of this process, and give extensive experimental evidence that the assumptions of our model are reasonable. We then characterize all possible knot or catenane products that arise from the most common substrates. We apply our model to tightly prescribe the knot or catenane type of previously uncharacterized data.

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Properties of a mutant Cre protein that alters the topological linkage of recombination products

Cited by 27 publications

References 14 publications

Isolation and characterization of intermediates in site-specific recombination.

Isolation and characterization of intermediates in site-specific recombination.

Growth inhibition and DNA damage induced by Cre recombinase in mammalian cells

Predicting Knot or Catenane Type of Site-Specific Recombination Products

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