Instability of chromosomal fragile sites is directly related to many cancers. Three types of fragile sites are generated under three different culture conditions: FRA3B, an aphidicolin-inducible fragile site, FRA11B, a folate-sensitive site, and FRA16B, a distamycin-A-inducible site, are involved in the formation of cancers. Compact chromatin structures and unusual DNA sequences have been found in fragile sites. Also, fragile sites display replication delay and can escape the ATR-dependent replication checkpoint. These observations provide an intriguing model for the nature of fragile sites, in which chromatin and DNA structures at these sites would pause the progress of the replication fork, and disrupt cell cycle checkpoint pathways to allow chromosomal rearrangement and viral integration, resulting in fragile site-specific tumorigenesis. Three goals are proposed: (1) Identify unique determinants for the fragile site-specific chromatin. Chromatin immunoprecipitation (CHIP) assays will be employed to identify epigenetic marks involved in these sites. Further, by reconstituting chromatin over all three fragile DNAs, fragile site-specific chromatin structure will be analyzed, and the essential components involved in the formation of fragile chromatin will be identified. (2) Establish cell cycle checkpoint pathways involved in the expression of fragile sites. Using CHIP assay and mutant cells created by RNA interference, the involvement of several cell cycle checkpoint proteins in the expression of fragile sites will be examined for their association with fragile DNA. (3) Determine cis- and trans-factors affecting fragile site instability by using an SV40 replication model system. By manipulating fragile DNA (length, sequences, replication direction, and location relative to replication origin) and cell cycle checkpoint components, fragile site instability (generation of break sites and changes in repeat length) will be evaluated. Replication intermediates containing fragile DNAs will also be characterized to provide direct information about replication delay of fragile sites. This proposal addresses how fragile sites lead to oncogenic lesions at three sequential steps. Therefore, these experiments will further advance knowledge about the nature of these fragile sites and their role in the formation of cancer, and also address fundamental biological questions, such as determinants of chromatin structure. ? ?
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