LS-1-P-5882 DNA repair studies in living cells

The maintenance of genome integrity is fundamental for proper cellular functions. Cells are continuously exposed to genotoxic factors, including UV irradiation or oxidative stress induced by pollutants. Therefore, faultless DNA repair is more than demanding for genome stability. Genotoxic stress generally leads to induction of DNA lesions that must be repaired in order to avoid deleterious chromosomal translocations. Therefore, in irradiated chromatin of living cells we analyze kinetics and appearance of proteins involved in DNA repair pathways or proteins recognizing the changes in radiation-caused chromatin conformation. For induction of DNA lesions we are using various sources of radiation, including UVA lasers or gamma-rays. From the view of various types of DNA lesions, we study the cell cycle dependent recruitment of selected proteins at radiation-damaged chromatin. An example represents PCNA protein according to which it is possible to recognize cells in the non-S-phase (Fig. 1A) and the S-phase (Fig. 1B) of the cell cycle. To distinguish G1 and G2 phases of the cell cycle, we are using HeLa-Fucci cellular system expressing RFP-cdt1 in the G1 phase, and GFP-geminin in the G2 phase of the cell cycle. For example, by this experimental approach we showed that coilin, a protein of Cajal bodies, has ability to recognize DNA lesions appearing in both G1 and G2 cell cycle phases. Our aim is also to study protein-protein or protein-DNA interactions and kinetics in locally induced DNA lesions of living cells after intervention to epi-genome.