Genome Instability at Common Fragile Sites. Updating the causes of their variability in different cell tissues. 2019. in: Eukaryotic DNA Replication & Genome Maintenance, Cold Spring Harbor Laboratory, September, 3 - 7, 2019, New York, USA.

Common Fragile Sites (CFSs) are chromosome regions prone to breaks or constrictions. CFSs are characterized by late replication timing mainly due to enrichment of A/T nucleotides which form secondary structures, to the paucity of active replication origins and to the presence of large genes which require long time to be completely transcribed and in which may occur the collision between replication and transcription processes that could lead to the formation of R-loop. An incomplete DNA duplication results in genomic instability. Hence, to understand the causes of their instability it is necessary to investigate the relationship between replication and fragility, in association with tissue specificity. Thus, we induced the expression of CFSs in stressful condition by using an inhibitor of DNA polymerase (aphidicolin) in MRC-5 and IMR-90, two human lung fibroblast cell lines respectively of 14 and 16 weeks old, in order to compare the cytogenetic localization of CFSs between the same tissue but derived from two distinct individuals, different genders and different stage of differentiation. The most expressed CFSs in both cell lines are 1p31.1 on chromosome 1 and 3q13.3 on chromosome 3. Moreover, we identify another interesting fragile site at 7q11.21-22. These sites have typical features of CFSs and in the most fragile region are present many large and late replicated genes. Furthermore, the percentages of the repetitive elements in each sites were compared with the non-fragile regions and the standard genomic sequences (Smit, 1999) showing no significative differences. For cytogenetic characterization of these fragile sites in fibroblasts, FISH (fluorescence in situ hybridization) technique was performed to delimit the boundaries of the fragile regions on chromosomes. Successively, it was determined the replication profiles of these three fragile sites in normal and in stressful conditions through combined analysis of FISH and immunofluorescence against BrdU (thymidine analogue) on interphase nuclei. For additional control, the same probes were used on lymphocyte metaphases and nuclei, in which these regions are not fragile, although display a non-canonical replication timing, indicating the presence of structural peculiarities that impair a normal replicative fork progression. The results show an incomplete replication of alleles at the end of S-phase under stress condition in both tissues, which raise fragility in fibroblasts, but not in lymphocytes, revealing a strict correlation between the expression of CFSs and tissue specificity. These outcomes suggest a prominent role of DNA replication in promoting a strong chromosomal instability.