Analysis of Restriction Enzyme-Induced DNA Double-Strand Breaks in Chinese Hamster Ovary Cells by Pulsed-Field Gel Electrophoresis: Implications for Chromosome Damage

1991 ◽  
Vol 128 (2) ◽  
pp. 150 ◽  
Author(s):  
Darlene D. Ager ◽  
John W. Phillips ◽  
Elma Abella Columna ◽  
Richard A. Winegar ◽  
William F. Morgan
Keyword(s):  
Restriction Enzyme ◽  
Gel Electrophoresis ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Pulsed Field Gel Electrophoresis ◽  
Double Strand Breaks ◽  
Chromosome Damage ◽  
Dna Double Strand Breaks ◽  
Ovary Cells ◽  
Strand Breaks

scholarly journals Illegitimate recombination induced by DNA double-strand breaks in a mammalian chromosome.

1994 ◽  
Vol 14 (9) ◽  
pp. 5794-5803 ◽  
Author(s):  
J W Phillips ◽  
W F Morgan
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Induced Mutations ◽  
Ovary Cells ◽  
Strand Breaks ◽  
Dna Double Strand Break ◽  
Aprt Gene

We examined DNA double-strand-break-induced mutations in the endogenous adenine phosphoribosyl-transferase (APRT) gene in cultured Chinese hamster ovary cells after exposure to restriction endonucleases. PvuII, EcoRV, and StuI, all of which produce blunt-end DNA double-strand breaks, were electroporated into CHO-AT3-2 cells hemizygous at the APRT locus. Colonies of viable cells containing mutations at APRT were expanded, and the mutations that occurred during break repair were analyzed at the DNA sequence level. Restriction enzyme-induced mutations consisted of small deletions of 1 to 36 bp, insertions, and combinations of insertions and deletions at the cleavage sites. Most of the small deletions involved overlaps of one to four complementary bases at the recombination junctions. Southern blot analysis revealed more complex mutations, suggesting translocation, inversion, or insertion of larger chromosomal fragments. These results indicate that blunt-end DNA double-strand breaks can induce illegitimate (nonhomologous) recombination in mammalian chromosomes and that they play an important role in mutagenesis.

Increased repair of γ-induced DNA double-strand breaks at lower dose-rate in CHO cells

2004 ◽  
Vol 82 (2) ◽  
pp. 125-132 ◽  
Author(s):  
Didier Boucher ◽  
Joëlle Hindo ◽  
Dietrich Averbeck
Keyword(s):  
Dose Rate ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Γ Irradiation ◽  
Ovary Cells ◽  
Strand Breaks

DNA double-strand breaks (DSBs) are highly cell damaging. We asked whether for a given dose a longer irradiation time would be advantageous for the repair of DSBs. Varying the γ-irradiation dose and its delivery time (0.05 Gy/min low dose-rate (LDR) compared with 3.5 Gy/min high dose-rate), confluent Chinese hamster ovary cells (CHO-K1) and Ku80 mutant cells (xrs-6) deficient in nonhomologous end-joining (NHEJ) were irradiated in agarose plugs at room temperature using a cesium-137 γ-ray source. We used pulsed-field gel electrophoresis (PFGE) to measure DSBs in terms of the fraction of activity released (FAR). At LDR, one third of DSBs were repaired in CHO-K1 but not in xrs-6 cells, indicating the involvement of NHEJ in the repair of γ-induced DSBs at a prolonged irradiation incubation time. To improve DSB measurements, we introduced in our PFGE protocol an antioxidant at the cell lysis step, thus avoiding free-radical side reactions on DNA and spurious DSBs. Addition of the metal chelator deferoxamine (DFO) decreased more efficiently the basal DSB level than did reduced glutathione (GSH), showing that measuring DSBs in the absence of DFO reduces precision and underestimates the role of NHEJ in the dose-rate effect on DSB yield.Key words: γ-irradiation, Chinese hamster ovary cells (CHO), nonhomologous end-joining (NHEJ), low dose-rate, deferoxamine.

Illegitimate recombination induced by DNA double-strand breaks in a mammalian chromosome

1994 ◽  
Vol 14 (9) ◽  
pp. 5794-5803
Author(s):  
J W Phillips ◽  
W F Morgan
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Induced Mutations ◽  
Ovary Cells ◽  
Strand Breaks ◽  
Dna Double Strand Break ◽  
Aprt Gene

We examined DNA double-strand-break-induced mutations in the endogenous adenine phosphoribosyl-transferase (APRT) gene in cultured Chinese hamster ovary cells after exposure to restriction endonucleases. PvuII, EcoRV, and StuI, all of which produce blunt-end DNA double-strand breaks, were electroporated into CHO-AT3-2 cells hemizygous at the APRT locus. Colonies of viable cells containing mutations at APRT were expanded, and the mutations that occurred during break repair were analyzed at the DNA sequence level. Restriction enzyme-induced mutations consisted of small deletions of 1 to 36 bp, insertions, and combinations of insertions and deletions at the cleavage sites. Most of the small deletions involved overlaps of one to four complementary bases at the recombination junctions. Southern blot analysis revealed more complex mutations, suggesting translocation, inversion, or insertion of larger chromosomal fragments. These results indicate that blunt-end DNA double-strand breaks can induce illegitimate (nonhomologous) recombination in mammalian chromosomes and that they play an important role in mutagenesis.

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