TARGETED BREAKAGE OF PARACENTROMERIC HETEROCHROMATIN INDUCES CHROMOSOMAL INSTABILITY

Current models suggest that genomic instability is crucial in the accu mulation of the multiple alterations required for tumorigenesis, Howev er, the nature of the initial damage responsible for the origin of gen omic instability remains poorly understood. In this investigation we d emonstrate that the nucleotide analog 2,6-diaminopurine (DAP) can be u sed to induce highly focused damage to the large blocks of paracentrom eric heterochromatin on chromosomes 1, 9 and 16, A large fraction of c ells exposed to DAP exhibit undercondensation of a and classical heter ochromatin which persists into metaphase, Subsequent chromosome breaka ge was observed for one of the target chromosomes by preferential excl usion of chromosome 16 fragments into micronuclei (P < 0.0001). The sp ecificity of DAP-induced chromosomal breakage enabled us to utilize it as a reagent to demonstrate that paracentromeric heterochromatin is a sensitive target for the induction of persistent genomic instability. We observed a 100-fold increase in mutagenesis affecting a chromosome 16 marker (APRT) compared with marker loci on chromosomes 17 (TK) or X (HPRT), We previously reported that APRT(-) mutants were recovered a t a high rate upon selection in DAP in a process involving recombinati onally mediated loss of heterozygosity that extends from the telomere to the boundary region of the paracentromeric heterochromatin. Karyoty pic analysis of DAP-resistant APRT- mutant clones demonstrated extensi ve genomic instability, particularly evidence of multiple and sequenti al events affecting chromosome 16. These data suggest that the heteroc hromatic breakage observed cytogenetically immediately following DAP e xposure is also responsible for the initiation of persistent genomic i nstability.