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Previous webinars > 3rd webinar 03/08/20223rd EDRA Webinar March. 8, 2022, from 4.30 to 6 pm CET
Dr Burkhard Jakob - Head of the Molecular Radiobiology & Imaging Group Biophysics, GSI, Germany www.gsi.de
Watching repair processes in real time - damage quality matters! In this webinar, I will discuss the application of live cell microscopy techniques to study the dynamics of the DNA damage response (DDR) and DNA repair processes after ionizing radiation and compare it to results from fixed cells. In our laboratory at the GSI Helmholtzzentrum für Schwerionenforschung, we are especially interested in the formation and processing of clustered DNA double-strand breaks as introduced by accelerated charged particles in comparison to sparsely ionising radiation (e.g. x-rays). Imaging of recruitment behaviour of different repair factors using beamline microscopy together with studies of protein binding and chromatin changes revealed differences in the DDR depending on the ionization density as well as for ionizing radiation in comparison to laser micro-irradiation. Especially combining different microscopy techniques can help us to understand the importance of the track structure and subnuclear dose distribution on repair and subsequent cellular endpoints in view of the enhanced biological effectiveness (RBE) of charged particles.
Short 15-min talks: 1. Hubert Fleury - University of Colorado Boulder, USA The APE2 nuclease is essential for alternative end-joining and survival of HR-deficient cells Alternative end-joining (altEJ) is a non-accurate pathway of DNA double strand break repair that relies on PARP1, XRCC1/Ligase 3 and the polymerase Theta. AltEJ is essential for survival of cells that are deficient for homologous recombination, and has therefore emerged as a powerful target to eliminate HR-deficient cancers. We used this synthetic lethality as a tool to identify potentially uncharacterized factors of altEJ, by performing genome-wide CRISPR-Cas9 screens in isogenic HR-proficient, BRCA1-KO, and PALB2-KO cells. We identified all the known altEJ factors, as well as ALC1, CIP2A and APEX2. We tested altEJ activity in telomere-fusion assays and at DNA reporters upon deletion of those genes, and found that the endo/exonuclease APE2 is essential for altEJ repair in both types of assays. We confirmed that APE2 is recruited to laser-induced micro-irradiation breaks, and further identified the domains of APE2 that are required for altEJ repair. Strikingly, the domains that are essential/dispensable for altEJ are also essential/dispensable for survival in HR-deficient cells, suggesting that the synthetic lethal interaction between Ape2 and HR is mainly due to Ape2's function in altEJ. Using biochemistry and innovative genomic assays, we are currently deciphering the molecular function of APE2 in this pathway. Because suppression of APE2 exquisitely kills HR-deficient cells, we believe it has a strong potential as a target against HR-deficient cancers.
2. Siham Zentout - Institute Genetics & Development of Rennes, CNRS, France HPF1 dependent histone PARylation drives DNA damage induced chromatin relaxation One of the earliest detectable events after DNA damage induction is the recruitment of PARP1 and its binding partner, HPF1 to sites of damage. Together, these proteins catalyze the addition of PAR chains specifically on serine residues to proteins in and around break sites, with the main acceptors being PARP1 and histones. While PARP1 dependent PARylation has been shown to drive chromatin relaxation immediately after DNA damage, the role of HPF1 in this process is unknown. In this current study, we aim to decipher the role of HFP1 in early DNA damage induces chromatin remodeling events. Using live-cell imaging approaches, we show that the recruitment of HPF1 to sites of damage relies on an interaction with the two terminal PARP1 residues, as recently described in crystal structures, and that in the absence of HPF1, DNA damage induced chromatin relaxation is greatly diminished. Furthermore, we are able to show that HPF1 dependent PARylation of histones and not PARP1 auto-PARylation drives this relaxation process. Finally, we show that loss of HPF1 reduces DNA repair efficiency by reducing the recruitment of repair factors to sites of damage. |
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