Sunday, October 16, 2005 3:00 PM-5:00 PM Exhibit Hall

(PP059) Radiation-induced chromatid breaks: are they formed by errors in chromatid deconcatenation?

Bryant, Peter*,1, Campbell, Elaine1, Comrie, Muriel1, 1 Bute Medical School, St Andrews, Fife, Scotland

ABSTRACT- Radiation-induced chromatid breaks: are they formed by errors in chromatid deconcatenation? Peter E Bryant, Elaine Cambell and Muriel Comrie Bute Medical School, University of St Andrews, St Andrews KY16 9TS, Scotland Chromatid breaks (CB) that originated in G2 phase cells in response to ionizing radiation are known to be caused by DNA double-strand breaks (DSB). However, the link between DSB and CB is obscure. For example abrogation of CB 'rejoining' by adenine arabinoside occurs under conditions where DSB rejoining is apparently unaffected. Confirmation of the role of DSB is provided by the finding that the frequency of CB is increased as expected in cells with mutations in genes involved in the NHEJ pathway, but also in cells with mutations in genes such as XRCC8 and ATM where bulk DSB rejoining is seemingly normal. These experimental results argue against the notion of a direct coupling of DSB and CB and hence do not support the classical breakage and reunion hypothesis for CB. For mammalian cells the induction of CB is linear with dose in several cell lines tested indicating a one-hit process is involved (i.e. presumably 1 DSB). We have been testing an entirely new concept for the formation of CB. We propose that the causative DSB is not directly involved in the formation of the CB and that a small fraction of DSB trigger errors (end mis-joining) in chromatin-loop deconcatenation by topoisomerase II. This idea was suggested to us by the effects of a catalytic topo II inhibitor (ICRF 193). This inhibitor causes a severe G2 blockade. However, cells that eventually escape the block and enter mitosis show multiple CB. We suggest in an analogous way radiation-induced CB are a consequence of disruption caused by the DSB resulting in end mis-joining during deconcatenation, and resulting in a loop rearrangement (inversion) with associated CB. Disappearance of CB occurs on completion of mis-joining. It is known that 16% of CB involve inter-chromatid rearrangements. Thus, up to 84% of CB could be caused by the above mechanism. To test this we have derived model cell lines containing unique DSB sites that can be incised by an endonuclease. The DSB site is highlighted by a plasmid-FISH probe and the signal amplified using tyramide-fluorescein. CB at such a unique site have indicated the presence of an inversion adjacent to the break-site.

Key words: chromatid break, DNA double-strand break, topoisomerase II, deconcatenation

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2005 RRS