Homologous recombination catalyzed by human cell extracts.

Two plasmids containing noncomplementing and nonreverting deletions in a bacterial phosphotransferase gene conferring resistance to neomycin (Neor) were incubated with human cell extracts, and the mixtures were used to transform recombination-deficient (recA-) Escherichia coli cells. We were able to obtain Neor colonies at a frequency of 2 X 10(-3). This frequency was 100 to 1,000 times higher than that obtained with no extracts. The removal of riboadenosine 5'-triphosphate, Mg2+, or deoxynucleoside triphosphates from the reaction mixture severely reduced the yield of Neor colonies. Examination of plasmid DNA from the Neor colonies revealed that they resulted from gene conversion and reciprocal recombination. On the basis of these results, we conclude that mammalian somatic cells in culture have the enzymatic machinery to catalyze homologous recombination in vitro.

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Rapid and apparently error-prone excision repair of nonreplicating UV-irradiated plasmids in Xenopus laevis oocytes

Molecular and Cellular Biology ◽

10.1128/mcb.10.7.3505-3511.1990 ◽

1990 ◽

Vol 10 (7) ◽

pp. 3505-3511

Author(s):

J B Hays ◽

E J Ackerman ◽

Q S Pang

Keyword(s):

Escherichia Coli ◽

Homologous Recombination ◽

Plasmid Dna ◽

Excision Repair ◽

Xenopus Laevis Oocytes ◽

Marked Contrast ◽

Repair Synthesis ◽

Error Frequency ◽

Repair Activity ◽

Good Agreement

Repair of UV-irradiated plasmid DNA microinjected into frog oocytes was measured by two techniques: transformation of repair-deficient (delta uvrB delta recA delta phr) bacteria, and removal of UV endonuclease-sensitive sites (ESS). Transformation efficiencies relative to unirradiated plasmids were used to estimate the number of lethal lesions; the latter were assumed to be Poisson distributed. These estimates were in good agreement with measurements of ESS. By both criteria, plasmid DNA was efficiently repaired, mostly during the first 2 h, when as many as 2 x 10(10) lethal lesions were removed per oocyte. This rate is about 10(6) times the average for removal of ESS from repair-proficient human cells. Repair was slower but still significant after 2 h, but some lethal lesions usually remained after overnight incubation. Most repair occurred in the absence of light, in marked contrast to differentiated frog cells, previously shown to possess photoreactivating but no excision repair activity. There was no increase in the resistance to DpnI restriction of plasmids (methylated in Escherichia coli at GATC sites) incubated in oocytes; this implies no increase in hemimethylated GATC sites, and hence no semiconservative DNA replication. Plasmid substrates capable of either intramolecular or intermolecular homologous recombination were not recombined, whether UV-irradiated or not. Repair of Lac+ plasmids was accompanied by a significant UV-dependent increase in the frequency of Lac- mutants, corresponding to a repair synthesis error frequency on the order of 10(-4) per nucleotide.

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Effect of double-strand breaks on homologous recombination in mammalian cells and extracts

Molecular and Cellular Biology ◽

10.1128/mcb.5.12.3331-3336.1985 ◽

1985 ◽

Vol 5 (12) ◽

pp. 3331-3336

Author(s):

K Y Song ◽

L Chekuri ◽

S Rauth ◽

S Ehrlich ◽

R Kucherlapati

Keyword(s):

Homologous Recombination ◽

Mammalian Cells ◽

Recombination Frequency ◽

Double Strand Breaks ◽

Base Pairs ◽

In Vitro System ◽

Strand Breaks ◽

Cell Extracts ◽

Nuclear Extracts

We examined the effect of double-strand breaks on homologous recombination between two plasmids in human cells and in nuclear extracts prepared from human and rodent cells. Two pSV2neo plasmids containing nonreverting, nonoverlapping deletions were cotransfected into cells or incubated with cell extracts. Generation of intact neo genes was monitored by the ability of the DNA to confer G418r to cells or Neor to bacteria. We show that double-strand breaks at the sites of the deletions enhanced recombination frequency, whereas breaks outside the neo gene had no effect. Examination of the plasmids obtained from experiments involving the cell extracts revealed that gene conversion events play an important role in the generation of plasmids containing intact neo genes. Studies with plasmids carrying multiple polymorphic genetic markers revealed that markers located within 1,000 base pairs could be readily coconverted. The frequency of coconversion decreased with increasing distance between the markers. The plasmids we constructed along with the in vitro system should permit a detailed analysis of homologous recombinational events mediated by mammalian enzymes.

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Effect of polyethylene glycol in plasmid DNA solution on transformation of CaCl2-treated Escherichia coli cells.

Agricultural and Biological Chemistry ◽

10.1271/bbb1961.48.657 ◽

1984 ◽

Vol 48 (3) ◽

pp. 657-662 ◽

Cited By ~ 1

Author(s):

Michio HIMENO ◽

Toshihiro SHIBATA ◽

Yoshio KAWAHARA ◽

Yuhji HANAOKA ◽

Tohru KOMANO

Keyword(s):

Escherichia Coli ◽

Polyethylene Glycol ◽

Plasmid Dna ◽

Escherichia Coli Cells

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Identification of a Reactivating Factor for Adenosylcobalamin-Dependent Ethanolamine Ammonia Lyase

Journal of Bacteriology ◽

10.1128/jb.186.20.6845-6854.2004 ◽

2004 ◽

Vol 186 (20) ◽

pp. 6845-6854 ◽

Cited By ~ 32

Author(s):

Koichi Mori ◽

Reiko Bando ◽

Naoki Hieda ◽

Tetsuo Toraya

Keyword(s):

Escherichia Coli ◽

Molecular Weight ◽

Protein S ◽

Amino Acid Residues ◽

Permeabilized Cells ◽

E Coli ◽

Cell Extracts ◽

Auxiliary Protein

ABSTRACT The holoenzyme of adenosylcobalamin-dependent ethanolamine ammonia lyase undergoes suicidal inactivation during catalysis as well as inactivation in the absence of substrate. The inactivation involves the irreversible cleavage of the Co-C bond of the coenzyme. We found that the inactivated holoenzyme undergoes rapid and continuous reactivation in the presence of ATP, Mg2+, and free adenosylcobalamin in permeabilized cells (in situ), homogenate, and cell extracts of Escherichia coli. The reactivation was observed in the permeabilized E. coli cells carrying a plasmid containing the E. coli eut operon as well. From coexpression experiments, it was demonstrated that the eutA gene, adjacent to the 5′ end of ethanolamine ammonia lyase genes (eutBC), is essential for reactivation. It encodes a polypeptide consisting of 467 amino acid residues with predicted molecular weight of 49,599. No evidence was obtained that shows the presence of the auxiliary protein(s) potentiating the reactivation or associating with EutA. It was demonstrated with purified recombinant EutA that both the suicidally inactivated and O2-inactivated holoethanolamine ammonia lyase underwent rapid reactivation in vitro by EutA in the presence of adenosylcobalamin, ATP, and Mg2+. The inactive enzyme-cyanocobalamin complex was also activated in situ and in vitro by EutA under the same conditions. Thus, it was concluded that EutA is the only component of the reactivating factor for ethanolamine ammonia lyase and that reactivation and activation occur through the exchange of modified coenzyme for free intact adenosylcobalamin.

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