RESTRICTION-ENDONUCLEASE-INDUCED DNA DOUBLE-STRAND BREAKS AND CHROMOSOMAL-ABERRATIONS IN MAMMALIAN-CELLS

Restriction endonucleases (RE) can be used to mimic and model the clas togenic effects of ionising radiation. With the development of improve d techniques for cell poration: electroporation and recently streptoly sin O (SLO), it has become possible more confidently to study the rela tionships between DNA double-strand breaks (dsb) of various types (e.g . blunt or cohesive-ended) and the frequencies of induced metaphase ch romosomal aberrations or micronuclei in cytokinesis-blocked cells. Alt hough RE-induced dsb do not mimic the chemical end-structure of radiat ion-induced dsb (i.e. the 'dirty' ends of radiation-induced dsb), it h as become clear that cohesive-ended dsb, which are thought to be the m ajor type of dsb induced by radiation, are much less clastogenic than blunt-ended dsb. It has also been possible, with the aid of electropor ation 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 concentratio ns persist and induce dsb over a period of many hours following treatm ent. Cutting of DNA by RE is thought to be at specific recognition seq uences (as in free DNA) although the frequencies of sites in native ch romatin available to RE is not yet known. DNA condensation and methyla tion are both factors limiting the numbers of available cutting sites. Relatively little is known about the kinetics of incision or repair o f RE-induced dsb in cells. Direct ligation may be a method used by cel ls to rejoin the bulk of RE-induced dsb, since inhibitors such as araA , araC and aphidicolin appear not prevent rejoining, although these in hibitors have been found to lead to enhanced frequencies of chromosoma l aberrations. 3-Aminobenzimide, the poly-ADP ribose polymerase inhibi tor 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 tr eated simultaneous with Pvu II and T4 ligase yielded lower levels of c hromosomal damage than in the WT parental line indicating that Pvu II induced dsb retain their ability to be blunt-end ligated inside the ce ll.