Effect of terminal non-homology on intramolecular recombination of linear plasmid substrates in Escherichia coli

Luisi-DeLuca, Cynthia; Kolodner, Richard D.

doi:10.1016/0022-2836(92)90682-a

Cited by 25 publications

(15 citation statements)

References 41 publications

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“…The RecE and A Red recombination pathways promote double-strand-break repair, which is unlikely to occur by a degradation and reannealing mechanism (53, 54). Analysis of linear dimer plasmid recombination promoted by the RecE pathway was also consistent with the involvement of other types of pairing reactions, like those promoted by RecA (19,47). These studies suggest that the RecE recombination pathway involves other types of homologous-pairing reactions besides exonucleolytic degradation and reannealing.…”

Section: Rdk Unpublished Results)supporting

confidence: 62%

Homologous pairing and strand exchange promoted by the Escherichia coli RecT protein.

Hall

Kolodner

1994

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

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RecT protein ofEscherichia coil promotes the formation of joint molecules between homologous linear double-stranded M13mp19 replicative-form bacteriophage DNA and circular single-stranded M13mpl9 DNA in the presence of exonuclease VIII, the recE gene product. The joint molecules were formed by a mechanis involving the pairing of the complementary strand of the linear double-stranded DNA substrate with the circular single-stranded DNA substrate coupled with the displacement of the noncomplementary strand. When the homologous linear double-stranded DNA substrate had homologous 3' or 5' single-stranded tails, thenRecT promoted homologous pairing and strand exchange in the absence of exonuclease VIII. Histone Hi could substitute for RecT protein; however, joint molecules formed in the presence of histone Hi did not undergo strand exchange. These results indicate that under the reaction conditions used, the observed strand exchange action is promoted by RecT and is not the result of spontaneous branch migration. These results are consistent with the observation that expression of RecE (exonuclease VII) and RecT substitutes for RecA in some recombination reactions in E. coil.

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Section: Rdk Unpublished Results)supporting

confidence: 62%

Homologous pairing and strand exchange promoted by the Escherichia coli RecT protein.

Hall

Kolodner

1994

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

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“…This degradation/reannealing/strand exchange mechanism explains how the presence of RecE and RecT can render some types of recombination RecA-independent (6). However, in vivo evidence indicates that the ends of a linear duplex DNA may not be involved directly in the initial pairing event, and it was suggested that internal duplex-duplex initiation events could be promoted by the RecE pathway (11,19). Also, it has been pointed out that DSBR, which clearly requires a pairing function, cannot occur simply by a degradation and reannealing mechanism (20).…”

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confidence: 99%

DNA Strand Invasion Promoted by Escherichia coli RecT Protein

Naveilhan

Kolodner

1998

Journal of Biological Chemistry

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The RecT protein of Escherichia coli is a DNA-pairing protein required for the RecA-independent recombination events promoted by the RecE pathway. The RecT protein was found to bind to both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) in the absence of Mg 2؉ . In the presence of Mg 2؉ , RecT binding to dsDNA was inhibited drastically, whereas binding to ssDNA was inhibited only to a small extent. RecT promoted the transfer of a single-stranded oligonucleotide into a supercoiled homologous duplex to form a D (displacement)-loop. D-loop formation occurred in the absence of Mg 2؉ and at 1 mM Mg 2؉ but was inhibited by increasing concentrations of Mg 2؉ and did not require a high energy cofactor. Strand transfer was mediated by a RecT-ssDNA nucleoprotein complex reacting with a naked duplex DNA and was prevented by the formation of RecT-dsDNA nucleoprotein complexes. Finally, RecT mediated the formation of joint molecules between a supercoiled DNA and a linear dsDNA substrate with homologous 3-single-stranded tails. Together these results indicate that RecT is not a helix-destabilizing protein promoting a reannealing reaction but rather is a novel type of pairing protein capable of promoting recombination by a DNA strand invasion mechanism. These results are consistent with the observation that RecE (exonuclease VIII) and RecT can promote RecAindependent double-strand break repair in E. coli.In Escherichia coli, the major recombination pathway requires the function of the RecA and RecBCD proteins (for reviews, see Refs. 1 and 2). The recombination and repair deficiencies in recB recC mutants can be suppressed by two types of mutations called sbcA and sbcB(sbcC) (3-5). The sbcA mutations map on the cryptic Rac prophage and induce the expression of the RecE and RecT proteins (for review, see Ref. 6). Recombination in recB recC sbcA mutants occurs by what is called the RecE pathway (7), which in many ways is similar to the bacteriophage Red pathway (6). A distinctive property of the RecE pathway is that it promotes RecA-independent recombination of circular plasmids as well as intramolecular recombination of linearized plasmid DNAs (8 -11) and also promotes RecA-independent double strand break repair (DSBR) 1 (12).These types of recombination events have been shown to require functional recE and recT genes (13, 14). The recE gene product is an ATP-independent exonuclease, also called exonuclease VIII (15). Exonuclease VIII degrades preferentially linear duplex DNA in the 5Ј to 3Ј direction, yielding 5Ј-mononucleotides and also degrades single-stranded DNA (ssDNA) at low rates (16). The recT gene product was found to bind to ssDNA and to promote the renaturation of complementary ssDNA in an ATP-independent fashion (17). Also, the RecT protein in combination with exonuclease VIII was shown to promote homologous pairing and strand exchange between a circular ssDNA and a linear duplex DNA. In this reaction, exonuclease VIII degraded the linear duplex to expose ssDNA that was then annealed by RecT to a compl...

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“…The increase was sequence-specific since the same amount of Chl did not affect the enzymatic activity of cells transfected with pHAP (Fig. 3) (21). To rule out the possibility that the sequence conversion is mediated by E. coli, direct DNA sequencing of a PCR-amplified fragment of Hirt DNA was carried out.…”

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confidence: 99%

Targeted gene correction of episomal DNA in mammalian cells mediated by a chimeric RNA.DNA oligonucleotide.

Yoon

Cole-Strauss

Kmiec

1996

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

210

156

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An experimental strategy to facilitate correction of single-base mutations of episomal targets in mammalian cells has been developed. The method utilizes a chimeric oligonucleotide composed of a contiguous stretch of RNA and DNA residues in a duplex conformation with double hairpin caps on the ends. The RNA/DNA sequence is designed to align with the sequence of the mutant locus and to contain the desired nucleotide change. Activity of the chimeric molecule in targeted correction was tested in a model system in which the aim was to correct a point mutation in the gene encoding the human liver/bone/kidney alkaline phosphatase. When the chimeric molecule was introduced into cells containing the mutant gene on an extrachromosomal plasmid, correction of the point mutation was accomplished with a frequency approaching 30%. These results extend the usefulness of the oligonucleotide-based gene targeting approaches by increasing specific targeting frequency. This strategy should enable the design of antiviral agents.Targeted correction of disease-related mutations or sitedirected inactivation of viral genes by homologous recombination would be a effective strategy for gene therapy. Unfortunately, homologous recombination in mammalian cells between a target gene and an exogenous DNA vector takes place at relatively low frequencies and is complicated by interference from an illegitimate recombination pathway that does not depend on sequence homology (1-5). An alternative approach involves targeted mutagenesis facilitated by triple-helixforming oligonucleotides coupled to cross-linking agents (6, 7). Such oligonucleotides have been used previously to change DNA sequences thereby altering gene expression but these approaches have been limited by the sequence restriction of the target that must consist of homopurine or homopyrimidine stretches (8, 9). Moreover, the generation of a specific type of mutation or correction has been difficult to achieve (6, 7).We have developed an experimental strategy to enable correction of single-base mutations of episomal sequences by using a chimeric oligonucleotide of unique design. This strategy evolved from in vitro studies on homologous recombination conducted with the RecA and Rec2 proteins (10-12). Analysis of the homologous pairing reaction promoted by the Rec2 protein of Ustilago maydis revealed that RNADNA hybrids were more active in homologous pairing reactions than corresponding DNA duplexes (10). Since pairing would appear to be the rate-limiting step during the gene targeting process (13), the overall frequency of recombination should be elevated if the number of pairing events is elevated. It was also discovered that joint molecule formation proceeded efficiently even when the ends of the hybrid were capped with double hairpin structures. These observations led us to a strategy for gene targeting in which vector design would exploit the natural recombinogenicity of RNA-DNA hybrids and would feature double-hairpin capped ends avoiding destabilization or de-The publicatio...

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Effect of terminal non-homology on intramolecular recombination of linear plasmid substrates in Escherichia coli

Cited by 25 publications

References 41 publications

Homologous pairing and strand exchange promoted by the Escherichia coli RecT protein.

Homologous pairing and strand exchange promoted by the Escherichia coli RecT protein.

DNA Strand Invasion Promoted by Escherichia coli RecT Protein

Targeted gene correction of episomal DNA in mammalian cells mediated by a chimeric RNA.DNA oligonucleotide.

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