Workpackage 1 – cells cultivation
In these studies, we would like to show that orchestrated epigenetic regulation of chromatin is highly responsible for reparation of DNA strand breaks.
Description of work
Task 1.1. In the first year, we shall prepare constructs of proteins with fluorescence tags. The efficiency of generation DSBs spatially restricted to selected nuclear volumes exposed to laser beam will be assessed by immunodetection of ?H2AX (our preliminary results) and MDC1 in microirradiated areas.
Several independent experiments will be performed during the first 20 min after microirradiation to reveal the laser input set producing the minimal energy necessary and sufficient to generate discernible local DSB area in every pre-sensitized cell.
Task 1.2. Dynamics of HP1 proteins will be followed in cells mono transfected with one of HP1 isoforms tagged to EGFP in irradiated but also non irradiated chromatin domains by FRAP and FLIP techniques in different time intervals. The proportion of HP1 proteins phosphorylated at Thr50 to the total HP1 will be detected by immunodetection after cell fixation at different time PI and by western blot using antibody to HP1? (?) and to phosphorylated forms of the proteins (anti- HP1? (?)Thr50P).
ICKC SB RAS
D 1.1 Methods of conjugation of proteins with fluorescence tags, and cells cultivation and treatment for further experiments;
D 1.2. Publications.