The Use of Restriction Endonucleases to Study the Mechanisms of Chromosome Damage

Morgan, William F.; Winegar, Richard A.

doi:10.1007/978-3-642-75682-5_8

Cited by 21 publications

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“…Restriction enzymes have been shown by both pulsed-field gel electrophoresis (3) and neutral filter elution (10) to be highly efficient at inducing DNA double-strand breaks within the mammalian genome once introduced into cultured mammalian cells. Their introduction into mammalian cells has been shown to increase cell killing, genomic rearrangements in the form of chromosome aberrations and gene amplification (6,29,37), and mutation frequencies at several genetic loci (11,32,45).In the present communication, we report the types of alterations that were induced in the APRT gene by blunt-end DNA double-strand breaks produced by PvuII, EcoRV, and Stul at different locations within the gene. Using three restriction enzymes allowed us to examine the various types of recombination events that a blunt-end DNA double-strand break can induce in the APRT gene and to determine whether the location of the double-strand break within the gene, or the base sequences flanking the double-strand break, has any effect on the mutation process.…”

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

Phillips¹,

Morgan²

1994

Mol. Cell. Biol.

110

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We examined DNA double-strand-break-induced mutations in the endogenous adenine phosphoribosyltransferase (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.In the mammalian genome, DNA double-strand breaks can occur during cellular processes (4,46) or as the result of exposure to DNA-damaging agents (51). DNA double-strand breaks can cause chromosomal rearrangements (1), which can lead to cell killing (13), mutagenesis (25), and cell transformation (5). To understand how DNA double-strand breaks lead to genetic rearrangements, it is important to understand the mechanisms of DNA double-strand-break rejoining and how these processes are carried out at the DNA and chromosomal levels.Studies of plasmid integration in a variety of mammalian cell types, along with simian virus 40 (SV40) recircularization studies with monkey cells, have shown that mammalian cells predominantly repair DNA double-strand breaks by endjoining mechanisms that do not require extensive homology between the molecules to be joined (39). Recombination between nonhomologous DNA substrates was first described for bacteria (15) and has been defined as illegitimate recombination (16). Most studies examining illegitimate recombination have used DNA substrates linearized with various restriction endonucleases to produce specific combinations of end structures. These substrates are cleaved in vitro and passed through various cellular and cell-free systems, and the rejoined products are studied. Recircularization studies of a linearized SV40 genome passed through CV 1 monkey cells, and plasmid rejoining studies with Xenopus laevis egg and human cell extracts, have shown that these end-joining processes frequently use overlaps of one to six complementary bases (36,44,48). The products of illegitimate recombination between segments of mammalian chromosomes have been studied by examination of translocation breakpoints and deletion and insertion junctions at various genetic loci (24,25 bination, the initial steps leading to the recombinational event and the mechanisms involved are not understood.To study the...

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confidence: 82%

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

Phillips¹,

Morgan²

1994

Mol. Cell. Biol.

110

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“…Two methods were used to internalize REs into CHO cells, namely, the glycerol method and electroporation (Johannes et al 1992, Morgan andWinegar 1990) . Treatment of cells with hypertonic glycerol solutions could have been assumed to change G. A .…”

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Localization of Chromosome Breakpoints Induced byAluI andBamHI in Chinese Hamster Ovary Cells Treated in the G₁Phase of the Cell Cycle

Folle

Obe

1995

International Journal of Radiation Biology

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Intact Chinese hamster ovary cells were exposed to the restriction endonucleases (REs) AluI or BamHI. In metaphase spreads from these cells, 300 breakpoints per RE were localized in G-banded chromosome type aberrations (dicentrics, translocations, rings, terminal and interstitial deletions). The majority of breakpoints induced by both REs were localized in G-light bands and showed a similar distribution of breakpoint clusters. RE digestion of metaphase spreads with AluI induced C-banding, and with BamHI G-banding. The data indicate that nuclease sensitive sites associated with active genes are mainly responsible for the distribution of breakpoints.

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“…Studying the role of dsb in chromosome aberration formation has been greatly facilitated by the use of restriction enzymes (Bryant 1984, Natarajan and Obe 1984, Morgan and Winegar 1990 . In contrast to ionizing radiations, which induce a plethora of lesion types in DNA, restriction enzymes induce a single type of lesion, the dsb .…”

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Modelling the Formation of Polycentric Chromosome Aberrations

Sachs

Yates

Tarver

et al. 1992

International Journal of Radiation Biology

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Exchange-type chromosome aberrations produced by ionizing radiation or restriction enzymes are believed to result from pairwise interaction of DNA double-strand breaks (dsb). In addition to dicentrics, such aberrations may include higher-order polycentrics (tricentrics, tetracentrics, etc.). We have developed computer programs that calculate the probability of the various polycentrics for a given average number of pairwise interactions. Two models are used. Model I incorporates kinetic competition between restitution, complete exchanges (illegitimate recombination events), and incomplete exchanges. Model II allows unrestituted breaks even if there is no recombination. The models were applied to experimental observations of aberrations produced in G1 Chinese hamster ovary cells after electroporation with the restriction enzyme PvuII, which produces blunt-end dsb. We found, experimentally and theoretically, that there was a maximum in the number and multiplicity of polycentrics per cell: beyond a certain PvuII concentration no additional or higher-order polycentrics were produced. Computer-generated relationships, which were remarkably similar for both models and for all values of the adjustable parameters, were found between dicentrics per cell and higher-order polycentrics per cell. Excellent agreement was found between the experimental observations and the consensus theoretical curve relating tricentrics per cell to dicentrics per cell. The observed number of higher polycentrics per cell for a given number of dicentrics per cell was somewhat larger than the consensus theoretical prediction. The observed number of centric rings per cell was markedly larger than the consensus theoretical value, presumably owing to intrachromosomal localization ('proximity effects'). The computer models also provided estimates for the adjustable parameters; for example, in model I the fraction of incomplete exchanges was found to be about 35%.

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The Use of Restriction Endonucleases to Study the Mechanisms of Chromosome Damage

Cited by 21 publications

References 29 publications

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

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

Localization of Chromosome Breakpoints Induced byAluI andBamHI in Chinese Hamster Ovary Cells Treated in the G₁Phase of the Cell Cycle

Modelling the Formation of Polycentric Chromosome Aberrations

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The Use of Restriction Endonucleases to Study the Mechanisms of Chromosome Damage

Cited by 21 publications

References 29 publications

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

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

Localization of Chromosome Breakpoints Induced byAluI andBamHI in Chinese Hamster Ovary Cells Treated in the G1Phase of the Cell Cycle

Modelling the Formation of Polycentric Chromosome Aberrations

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Localization of Chromosome Breakpoints Induced byAluI andBamHI in Chinese Hamster Ovary Cells Treated in the G₁Phase of the Cell Cycle