Early events in DNA double strand break repair after damage induction with a laser microbeam
In the present thesis DNA double strand break (DSB) induction and repair are analysed after laser-microbeam irradiation. Live cell imaging of DNA repair proteins fused to Green Fluorescent Protein (GFP) as well as immunofluorescent detection of endogenous protein are used. Laser induced DNA damage, detected by gamma-H2AX foci staining depend on a subtle combination of used laser pulse wavelength, pulse energy and dose. The recruitment times of repair proteins depend inverse linearly on laser pulse energy. By extrapolation to zero, the recruitment time at biological relevant conditions is calculated. Interestingly, considerable spatial dynamics of the foci is found. Two neighbouring foci even can fuse within ~20 min. Recruitment time comparison of molecules representing early and late Non-Homologous end Joining (NHEJ), Homologous Recombination Repair (HRR) and the Mre11-Rad50-NBS1 (MRN) complex reveals that the whole NHEJ machinery is assembled to DSBs within 1 min. Recruitment of latest NHEJ factor (XRCC4) is faster than NBS1 and is not directly influenced by the absence of NBS1. XRCC4 persists at DSBs longer in the G1 cell cycle phase than in G2 where the replacement of NHEJ by HRR molecules occurs. Rad51 is recruited when XRCC4 is released with the complementary kinetics. DNA-PKcs phosphorylation at two sites, known to facilitate DNA end processing, occurs between the recruitment of NHEJ and HRR. A new modification of Comet-assay technique the Immunofluorescent Comet-assay (IFCA) is developed in this work for direct visualisation of DSBs in single cells. IFCA uses the immunofluorescent detection of histone H1 in neutral and alkaline Comet-assay, and enables simple and clear visualisation of details in the comet tail, which are hardly detectable by conventional DNA staining dyes such as SYBR Green. Using IFCA, the fragment size at the end of the neutral comet tail is determined for the first time.