Interstitial deletions and intrachromosomal amplification initiated from a double-strand break targeted to a mammalian chromosome

Interstitial deletions of tumour suppressor genes and amplification of 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‐ Sce I nuclease to introduce a localized double‐strand break (DSB) in a mammalian chromosome carrying its target sequence, we demonstrate here that both types of mutations can be initiated by non‐conservative DSB repair pathways. In our system, I‐ Sce I activity dissociates a transfected gpt gene from its promoter, allowing the isolation of gpt − clones. Our results show that intrachromatid single‐strand annealing events occur frequently, giving rise to interstitial deletions not accompanied by other chromosomal rearrangements. We also observed that, when present in the cells, extrachromosomal DNA molecules are integrated preferentially at the broken locus. Taking advantage of the insertion of the I‐ Sce I recognition sequence telomeric to and close to the dihydrofolate reductase gene, we show that a less frequent outcome of I‐ Sce I activity is the initiation of cycles of intrachromosomal amplification of this marker, from breaks at a site merging with the enzyme target.