Speaker
Dr
Sandro Conrad
(Technical University of Darmstadt)
Description
The major repair pathways for DNA double-strand breaks (DSBs) are non-homologous end-joining (NHEJ) and homologous recombination (HR). NHEJ is active in all cell cycle phases and repairs DSBs with fast kinetics. It represents the major repair pathway in G1 and G2. In contrast, HR is the main repair pathway for replication-associated DSBs in S phase and also repairs a subfraction of DSBs in G2. In G1 and G2 a small fraction of DSBs, representing those located in heterochromatic regions, is repaired with slow kinetics. This slow repair component represents HR in G2 phase but an NHEJ process in G1.
Deficiency of the endonuclease Artemis leads to a significant repair defect after X-irradiation in the slow repair component in G1 and G2. In G2, this Artemis dependent defect is associated with HR-mediated DSB repair, but the precise role of Artemis remains unclear. One model is that Artemis endonuclease activity processes secondary or cruciform structures which preferentially arise during the resection of DSBs in heterochromatin. To test this model we depleted CtIP, a factor necessary for DSB end resection and HR, in an Artemis deficient background and irradiated the cells with X-rays. Our results indicate that CtIP depletion completely rescues the Artemis repair defect in G1 and G2, suggesting that in the absence of CtIP DSBs are repaired independently of Artemis by NHEJ.
In contrast to X-rays, irradiation with high-LET particles leads to a pronounced DSB-repair defect in Artemis-downregulated G1 and G2 cells. The repair defect was more severe compared to Artemis-depleted cells irradiated with X-rays; almost all DSBs remained unrepaired up to 72h post IR and the lack of repair was independent of the chromatin status of the DSBs. Interestingly, depletion of CtIP alone also induced a pronounced repair defect after high-LET irradiation in G1 and G2, which is in stark contrast to the irradiation with X-rays.
This leads to the assumption that almost all DSBs induced by high-LET radiation undergo resection by CtIP irrespective of the cell cycle phase and their chromatin-status. Furthermore, co-depletion of CtIP and Artemis revealed that CtIP-depletion cannot rescue the Artemis-repair defect after high-LET irradiation. In summary, almost all DSBs induced by high-LET irradiation undergo resection by CtiP and further processing by Artemis, irrespective of their chromatin status; in G2-cells these resected and processed DSB are repai
Primary author
Dr
Sandro Conrad
(Technical University of Darmstadt)
Co-authors
Dr
Gisela Taucher-Scholz
(GSI Helmholzzentrum für Schwerionenforschung)
Prof.
Markus Löbrich
(Technical University of Darmstadt)
Dr
Olivia Barton
(Technical University of Darmstadt)
