Alterations in DNA conformation and histone phosphorylation resulting from double-strand breaks induced in HeLa cells – a spectroscopic and immunostaining approach

Abstract

All living cells are subjected daily to several thousands of DNA lesions of various origins. DNA damage leads to genetic material disorders that may have serious consequences, including cell apoptosis or mitotic death. The most dangerous DNA lesions are double-strand breaks (DSBs) accompanied by modifications of the DNA chemical structure. Here, we focus on DNA conformational transitions caused by DSBs formation induced by a genotoxic drug, bleomycin (BLM). The response of cells to this anticancer agent treatment was monitored with synchrotron radiation infrared micro-spectroscopy (SR-FTIR) and confocal laser scanning microscopy (CLSM). Obtained data were analyzed using multivariate data analysis methods. Depending on BLM concentrations, either DNA repair or apoptosis was induced. The applied methodology enabled tracking of molecular modifications on those processes, such as conformational transition from B-DNA to A-DNA and increased expression of proteins within the cell nuclei resulting from the activation of repair processes. Finally, we present a novel approach to determine the rate of chromatin damage based on computational analysis of the histone H2AX phosphorylation immunoassay.