Chromosomal fragile sites are specific loci which are especially susceptible to forming breaks or rearrangements when cells are cultured under conditions that inhibit DNA replication. The FRA3B at 3p14.2 is the most common aphidicolin-inducible fragile site. Band 3p14 may also contain a tumor suppressor gene, as evidenced by deletions and allele loss found in a variety of tumors. In the current funding period, we have cloned and characterized FRA3B, the first common fragile site to be isolated, and have begun to examine the mechanism for fragility. By inducing fragile sites and transfecting pSV2neo DNA that integrated into the FRA3B, we isolated DNA sequences flanking the integrated plasmid. We constructed a 100 kb genomic contig, and have demonstrated that these sequences are susceptible to breakage and recombination. In addition, we have found that deletions occur within FRA3B in human tumors. We have determined that FRA3B is late replicating, and that aphidicolin induces a further delay in the timing of replication, and that some cells enter G2 having failed to complete replication. Our results, together with those of other investigators, have revealed that FRA3B differs from the cloned rare fragile sites. First, instability occurs over at least a 500 kb region. Second, sequence analysis has not revealed trinucleotide repeats characteristic of the rare fragile sites. The molecular basis for instability at common fragile sites is unknown, but induction appears to involve the interruption of normal DNA synthesis. We have hypothesized that fragile sites are regions of DNA whose replication is unusually sensitive to interference, and that the visible chromosome breaks and gaps are due to unreplicated DNA. Moreover, we have proposed that transcription and late timing of replication may contribute to the instability of the FRA3B. The major goal of this proposal is to determine the mechanism(s) of fragile site expression and genetic instability. Specifically, we propose (1) to determine the role of DNA replication in fragile site expression, (2) to determine the role of gene transcription, and the potential interactions of gene transcription and the timing of replication in fragile site expression, and (3) to identify the specific sequences required for the manifestation of genetic instability. Genomic instability is a frequent characteristic of tumor cells, and common fragile sites may be particularly sensitive to such instability. We believe that the mechanisms involved in rearrangements at fragile sites are likely to apply to other sites of rearrangements in cancer, thus, our studies may lead to important insights into the pathogenesis of human tumors.
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