Restriction-endonuclease-induced DNA double-strand breaks and chromosomal aberrations in mammalian cells.
Restriction endonucleases (RE) can be used to mimic and model the clastogenic effects of ionising radiation. With the development of improved techniques for cell poration: electroporation and recently streptolysin O (SLO), it has become possible more confidently to study the relationships between DNA double-strand breaks (dsb) of various types (e.g. blunt or cohesive-ended) and the frequencies of induced metaphase chromosomal aberrations or micronuclei in cytokinesis-blocked cells. Although RE-induced dsb do not mimic the chemical end-structure of radiation-induced dsb (i.e. the 'dirty' ends of radiation-induced dsb), it has become clear that cohesive-ended dsb, which are thought to be the major type of dsb induced by radiation, are much less clastogenic than blunt-ended dsb. It has also been possible, with the aid of electroporation or SLO to measure the kinetics of dsb in cells as a function of time after treatment. These experiments have shown that some RE (e.g. Pvu II) are extremely stable inside CHO cells and at high concentrations persist and induce dsb over a period of many hours following treatment. Cutting of DNA by RE is thought to be at specific recognition sequences (as in free DNA) although the frequencies of sites in native chromatin available to RE is not yet known. DNA condensation and methylation are both factors limiting the numbers of available cutting sites. Relatively little is known about the kinetics of incision or repair of RE-induced dsb in cells. Direct ligation may be a method used by cells to rejoin the bulk of RE-induced dsb, since inhibitors such as araA, araC and aphidicolin appear not prevent rejoining, although these inhibitors have been found to lead to enhanced frequencies of chromosomal aberrations. 3-Aminobenzimide, the poly-ADP ribose polymerase inhibitor is the only agent that has so far been shown to inhibit rejoining of RE-induced dsb. Data from the radiosensitive xrs5 cell line, where chromosomal aberration frequencies are higher after RE treatments than in their normal parental CHO line, indicates that the xrs dsb repair pathway is involved in the repair of these dsb. We found that cells treated simultaneous with Pvu II and T4 ligase yielded lower levels of chromosomal damage than in the WT parental line indicating that Pvu II induced dsb retain their ability to be blunt-end ligated inside the cell.
Bryant
P E
PE
School of Biological and Medical sciences, University of St. Andrews, Fife, Scotland, UK.
Johnston
P J
PJ
D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D002869:Chromosome Aberrations; D006224:Cricetinae; D004247:DNA / Q000235:genetics* / Q000378:metabolism / Q000528:radiation effects; D004249:DNA Damage; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D007700:Kinetics; D008322:Mammals; D014965:X-Rays
D004247:DNA; D004262:DNA Restriction Enzymes