Modification of nuclear DNA by infrared femtosecond laser pulses

Abstract

DNA damage is one of the most severe insults cells have to cope with. To identify the damage-associated modifications of DNA is fundamental for understanding the mechanisms of DNA repair that lead to the maintenance of genome stability. Many key factors in damage recognition have been identified so far, but their exact interaction with DNA remains unclear. Femtosecond pulsed near-infrared lasers are a suitable tool to generate specific DNA damage in live cells and thus gain insights into the cellular recognition and response to DNA damage. In our laboratory, a near-infrared femtosecond pulsed fiber laser source with an emitting wavelength of 775 nm is coupled into a confocal laser scanning microscope in order to generate and visualize DNA damage. This wavelength-dependent non-linear process, which is based on multiphoton absorption, can be performed within the nucleus at a high spatial resolution. To determine the focal volume for efficient photodamaging processes, a fluorescent polymer slide was developed that allows measuring and calculating the spotsize by means of bleaching events. Further analysis of the effect of 775 nm on nuclear DNA included the establishment of new protocols for the detection of direct DNA strand break markers. The TUNEL assay was adapted to a single-cell level and together with the immunocytochemical labeling of Ku80 used to detect strand breaks at sites of laser-induced DNA damage. First approaches in live-cell imaging using the DNA repair factor XRCC1 demonstrated a power-dependent recruitment to damaged sites. Additionally, the newly established reporter cell line stably expressing eGFP-XRCC1 facilitates experimental handling and can easily be utilized to measure aberrations of the microirradiation system via a fast biological response. Time-lapse monitoring of the PAR turnover and recruitment kinetics of different PARP1 variants upon damage induction complete the comprehensive biological characterization of damage induction at 775 nm. The chromatin architectural protein DEK is known to be involved in various cellular processes including DNA repair and replication. It binds preferentially to cruciform DNA and heterochromatic regions. Previous studies demonstrated nuclear release of DEK in response to replication stress, which could not be reproduced in this study. To identify endogenous changes of DEK’s localization within the nucleus, a U2-OS cell line was established where WT DEK is replaced with an eGFP-tagged variant. In these cells, cell cycle-dependent focal accumulations of eGFP-DEK could be observed for the first time. Their time-window of appearance was identified as late S-phase based on colocalization studies with the replication factor PCNA. The defined timing of these DEK bodies even in unstressed conditions might indicate an important role of DEK during replication and further experiments will identify possible interaction partners and modulators of DEK foci formation.