Téma: Molekulární mechanismy a biomarkery I
Číslo abstraktu: 019
Common fragile sites (CFS) are evolutionary conserved hardreplicable genomic loci present also in human genome. CFS are prone to breakage especially upon replication stress most likely due to complicated secondary DNA structure resulting from specific nucleotide sequences. Increased manifestation of various lesions at CFS was reported for precancerous lesions as well as tumour tissue. We performed a proteomic screening for detection of proteins interacting with secondary DNA structure typically formed at CFS. Among others, we identified XPC protein and briefly characterized its role in CFS maintenance.
Replication stress was induced by aphidicolin (APH) in HeLa and U2OS cells. Part of the common fragile site FRA16D was synthetized and used as the DNA affinity ligand. Proteins selectively bound to the structure were identified and quantified by SILAC proteomic approach. The role of XPC protein in CFS stability was further characterized by molecular biology methods, e.g. immunofluorescence and western blotting.
Results and conclusions
We identified several proteins specifically binding to the synthetized FRA16D sequence upon APH-induced replication stress, including previously described WRN (Werner helicase). One of the hits, XPC protein, was chosen for further characterization because its role in CFS stability has not been reported yet. We observed attenuated activation of checkpoint response in XPC silenced cells upon replication stress. The obtained results indicate that the problematic genomic loci such as CFS are not properly recognized, nor repaired if cell deficient in functional XPC is exposed to replication stress. Therefore, in mitosis such regions are more likely converted to breaks with deleterious consequences for the cell.
Datum přednesení příspěvku: 2. 12. 2015