The replication of the two hotspots of breakage located within the human common fragile site FRA11D occurs in mid to late S phase : a preliminary study

Abstract

Cancer is a genetic disease characterized by the transformation of normal cells into malignant cells through uncontrollable divisions. Genomic instability, a key feature of genomic regions called fragile sites (FS), has been shown to be a hallmark of cancer. FS are specific DNA loci that show propensity to form gaps and breaks on metaphase chromosomes following DNA replication stress. Common Fragile Sites (CFS) are present in all individuals and are mainly induced by Aphidicolin, a specific DNA polymerases alpha and delta inhibitor. Calyculin A, which triggers premature chromosome condensation at any phase of the cell cycle, also induces CFS. The causes of their fragility are not yet fully deciphered; several causes are so far described such as replication fork stalling, paucity in initiation events, and late or slow replication. This latency might be due to CFS forming secondary structures, which upset the progression of the replication fork. In our work, we propose to investigate the replication timing at the CFS FRA11D which is located within the chromosomal band 11p14.2. Therefore, we selected from CHORI BACPAC two BAC clones, RP11-283H3 and RP11-1L12, each containing a specific sequence spanning a hotspot within FRA11D. We have tested with PCR, extracted, labeled and purified the BACs. These BACs were used as probes in FISH performed on lymphocytes cultured in the presence of bromodeoxyuridine, and treated with 60 ng/ml of calyculin A. The replication timing of these fragile regions has been compared to two controls, RP11-148G19 and RP11-3I14 with early and late replicating pattern respectively. Our data reveal that these two hotspots replicate in mid to late S phase. This finding provides a better understanding of the origin of genomic instability at the level of FRA11D observed in several genetic diseases.