Chromosome Aberrations Induced by Restriction Endonucleases

Obe, Günter; Vasudev, V.; Johannes, Christian

doi:10.1007/978-3-642-72802-0_13

Cited by 5 publications

(4 citation statements)

References 26 publications

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“…This study clearly shows that DNA double-strand breaks are the lesions that give rise to chromosome aberrations. This confirms the work of others extrapolating from ionizing radiations (6,34), from the use of single-strand-specific Neiurospora endonuclease to convert a single-strand break into a double-strand break (19,21,28), and from the addition of purified restriction endonucleases to permeabilized cells (3,20,22,33). The nature of the lesion leading to SCE formation remains a mystery.…”

Section: Discussionsupporting

confidence: 84%

“…A restriction endonuclease makes a double-strand break and few or no other perturbations in DNA. In the past, when commercially available restriction enzymes were introduced into permeabilized Chinese hamster cells, increased frequencies of SCEs (18) and chromosome aberrations (3,20,22,33) were observed. These studies, however, were hampered by (i) the necessity to permeabilize cells to permit access of the endonuclease yet keep the cells viable for cytogenetic assays, (ii) the lack of control over how much, if any, enzyme went into the cell, and (iii) the inability * Corresponding author.…”

mentioning

confidence: 99%

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Inducible expression and cytogenetic effects of the EcoRI restriction endonuclease in Chinese hamster ovary cells.

Morgan¹,

Fero²,

Land³

et al. 1988

Mol. Cell. Biol.

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The cytogenetic endpoints sister chromatid exchange (SCE) and chromosome aberrations are widely used as indicators of DNA damage induced by mutagenic carcinogens. Chromosome aberrations appear to result directly from DNA double-strand breaks, but the lesion(s) giving rise to SCE formation remains unknown. Most compounds that induce SCEs induce a spectrum of lesions in DNA. To investigate the role of double-strand breakage in SCE formation, we constructed a plasmid that gives rise to one specific lesion, a staggered-end ("cohesive") DNA double-strand break. This plasmid, designated pMENs, contains a selectable marker, neo, which is a bacterial gene for neomycin resistance, and the coding sequence for the bacterial restriction endonuclease EcoRI attached to the mouse metallothionein gene promoter. EcoRI recognizes G | AATTC sequences in DNA and makes DNA double-strand breaks with four nucleotides overhanging as staggered ends. Cells transfected with pMENS were resistant to the antibiotic G418 and contained an integrated copy of the EcoRI gene, detectable by DNA filter hybridization. The addition of the heavy metal CdSO4 resulted in the intracellular production of EcoRI, as measured by an anti-EcoRI antibody. Cytogenetic analysis after the addition of CdSO4 indicated a dramatic increase in the frequency of chromosome aberrations but very little effect on SCE frequency. Although there was some intercellular heterogeneity, these results confirm that DNA double-strand breaks do result in chromosome aberrations but that these breaks are not sufficient to give rise to SCE formation.Most mutagenic carcinogens induce sister chromatid exchanges (SCEs), chromosome aberrations, or both in eucaryotic cells. Although there is evidence that DNA doublestrand breaks lead to the formation of chromosome aberrations (3,6,(19)(20)(21)34), the precise lesion(s) and the mechanisms involved in the formation of SCEs are unknown. The relationship between a particular DNA lesion and its biological effect is often obscured by the fact that most mutagenic carcinogens induce a spectrum of lesions, the proportions of which vary in different cell types. For example, ionizing radiations induce single-and doublestrand breaks and make numerous base modifications (31), UV light generates cyclobutane and (6-4) pyrimidine-pyrimidone dimers plus a host of minor photoproducts (24), and alkylating agents alkylate both purines and pyrimidines in different ratios depending on the compound used (29). Ideally, to define the range of cytogenetic effects due to particular lesions, a specific class of lesions would be induced in cellular DNA in vivo, and the cytogenetic endpoint in question would be examined.Bacterial restriction endonucleases recognize specific, rather short sequences of DNA as binding sites. Binding is followed by either blunt-end or cohesive-end DNA cleavage at the recognition site itself or elsewhere, depending on the enzyme. A restriction endonuclease makes a double-strand break and few or no other perturbations in DNA. In the past, when ...

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Section: Discussionsupporting

confidence: 84%

mentioning

confidence: 99%

Inducible expression and cytogenetic effects of the EcoRI restriction endonuclease in Chinese hamster ovary cells.

Morgan¹,

Fero²,

Land³

et al. 1988

Mol. Cell. Biol.

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“…Revell ratios do not hold for S-cells, even for those late cells where most of the chromatin has completed replica-The efficiency of restriction endonucleases, introduced into cells, in producing S-independent aberrations qualitatively identical to those produced by radiation [Obe et al, 1985;Bryant, 1988Bryant, , 1990Morgan and Winegar, 19901, and the recent demonstration that radiation-induced and RE introduced breaks can interact [Tanzarella et al, 19901. All of these point to the fundamental importance of this lesion.…”

Section: Challenge To Classic Theory: the Exchange Theorymentioning

confidence: 94%

“…Such cells will contribute to lethality, but nucleases into cells, agents that produce double-strand breaks (DSB) at specific sequences, also produce aberrations at all stapes of the cell cycle [Bryant, 1984[Bryant, , 1988[Bryant, . 1990Obe et al, 1985;Natarajan and Obe, 1984;Morgan and Winegar, 19901. DSB are now recognised as a most important lesion in many radiobiological effects, and interpretations of most end-points have been given based upon them [Bender et al.…”

Section: Importance Of Whole Chromosomesmentioning

confidence: 98%

Update on target theory as applied to chromosomal aberrations

Savage

1993

Environ and Mol Mutagen

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The early radiobiologists, who developed target theory to explain their results, considered the chromosome "target" as a visible thread that could be physically broken by ionizing radiation. Most of the broken ends restituted, but those that did not were free to wander about and, within limits, could rejoin with any other broken end they happened to contact to form structural aberrations. Failing this, they could remain to be seen as "open" breaks at the subsequent metaphase. These ideas, and their inevitable consequences, still form the basis for much modern thinking, even though we now known that the structure of the chromosome, and of the interphase nucleus, are very much more complicated than the originators of the theory envisaged. Current understanding of chromosomes at the molecular level and the varied responses a cell can mobilize when damage is introduced, raise again the question, Can we still think in terms of simple targets? Some of the experimental observations and suggestions made since those early days are reviewed, and the application of target theory to the three theories of aberration origins (Classic, Exchange, Recombination) is briefly discussed.

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