INTERSTITIAL DELETIONS AND INTRACHROMOSOMAL AMPLIFICATION INITIATED FROM A DOUBLE-STRAND BREAK TARGETED TO A MAMMALIAN CHROMOSOME

Interstitial deletions of tumour suppressor genes and, amplification o f oncogenes are two major manifestations of chromosomal instability in tumour cells, The development of model systems allowing the study of the events triggering these processes is of major clinical importance, Using the properties of the I-SceI nuclease to introduce a localized double-strand break (DSB) in a mammalian chromosome carrying its targe t sequence, we demonstrate here that both types of mutations can be in itiated by non-conservative DSB repair pathways. In our system, I-SceI activity dissociates a transfected gpt gene from its promoter, allowi ng the isolation of gpt(-) clones, Our results show that intrachromati d single-strand annealing events occur frequently, giving rise to inte rstitial deletions not accompanied by other chromosomal rearrangements , We also observed that, when present in the cells, extrachromosomal D NA molecules are integrated preferentially at the broken locus, Taking advantage of the insertion of the I-SceI recognition sequence telomer ic to and close to the dihydrofolate reductase gene, we show that a le ss frequent outcome of I-SceI activity is the initiation of cycles of intrachromosomal amplification of this marker, from breaks at a site m erging with the enzyme target.