konn-te dagegen nachgewiesen werden, dass die schnelle Reparatur über das NHEJ er-folgt, während die langsame Kinetik durch die HR gekennzeichnet ist. Weitere Unter-suchungen zeigten, dass Artemis und ATM sowohl in die langsame Komponente der Reparatur in der G1- als auch in der G2-Phase involviert sind. Durch die Etablierung HR-spezifischer Nachweismethoden konnte für Artemis und ATM in der G2-Phase eine Beteiligung bei der HR aufgezeigt werden, während sie in der G1-Phase einen Weg des End-Joinings unterstützen. Die Artemis- und ATM-Abhängigkeit in der G2-Phase wird durch die Anresektion der DSBs, die von CtIP vermittelt wird, herbeige-führt. Überraschenderweise zeigte sich, dass auch in der G1-Phase die Anresektion der DSBs die Artemis- und ATM-Abhängigkeit hervorruft. Möglicherweise führt die Resektion der DSBs zur Ausbildung von sekundären Strukturen, die vor der Repara-tur beseitigt werden müssen. Da für Artemis eine Endonukleaseaktivität beschrieben ist, kann angenommen werden, dass Artemis für die Prozessierung der Sekundär-strukturen vonnöten ist.
Einblicke zu der Funktion von ATM bei der langsamen Reparaturkomponente lieferte die Tatsache, dass DSBs, die in der G2-Phase über die HR und in der G1-Phase über einen End-Joining-Weg repariert werden, im Heterochromatin lokalisiert sind.
Da für die Proteinkinase ATM beschrieben wurde, dass es den Heterochromatin-bildenden Faktor Kap1 phosphoryliert und so zur Änderung des Kap1-abhängigen Heterochromatinstatus beiträgt, lässt sich vermuten, dass ATM bei der lokalen Öff-nung des Heterochromatins beteiligt ist, die für eine erfolgreiche Reparatur strahlen-induzierter DSBs essentiell ist. Dabei scheint die ATM-abhängige Änderung des Heterochromatinstatus für die Reparatur der resektierten DSBs von besonderer Be-deutung zu sein.
Im Rahmen dieser Arbeit konnte die Kontrolle des G2/M-Checkpoints näher beleuch-tet werden. Zudem konnten Einblicke in das komplexe Reparaturverhalten in der G2- und G1-Phase gewonnen werden. Dabei stand die langsame Reparatur im Vorder-grund, die als Folge der Lokalisation im Chromatin bzw. der Anresektion der DSBs abläuft und von Artemis- und ATM-abhängig ist. Diese Erkenntnisse helfen dabei, die Reparatur besser zu verstehen und bieten viele Ansatzpunkte für zukünftige Arbei-ten.
This work is engaged in two different questions. The first part of this work deals with the effect of the tumor suppressor p53 on the G2/M transit. To investigate how cells with different p53 status are responding to ionizing radiation (IR) the induced G2/M checkpoint is analyzed by biochemical methods. It is shown, that p53 deficient cells have normal G2/M checkpoint induction after irradiation with 1 Gy, but cells are not released from the G2 arrest any more. So p53 seems to be important for the main-tenance of the G2 arrest. This function depends on the residual damage level. In contrast to this results irradiation with 12 Gy leads to a release from the G2 arrest.
The prolonged G2 arrest after the application of 1 Gy takes center stage in the further process of this work. The analysis of the repair ability of p53 deficient cells reveals that the repair capacity is not influenced and could be excluded as the reason for the prolonged G2 arrest. Instead it seems that p53 act upon the level of the DNA dam-age response checkpoint kinases Chk1/Chk2 whereby the cells abrogate the G2 ar-rest despite residual DNA damage after induction of a small amount of DSBs. A final conclusion of the exact role of p53 at the G2/M checkpoint is not possible yet. There-fore further examinations are required. From the results of the G2/M-checkpoint measurement we can conclude that p53 is important in the mitosis phase after irradi-ation and promotes the transit of mitotic cells with damaged DNA to the G1 phase.
These results reveal a new function of p53 in the cell cycle progression after inducing DNA damage with IR. The exact mechanism of p53 during mitosis needs to be study.
The second part of this work deals with the slow repair component in the G1 and G2 phase. Only a sub fraction of IR-induced DSBs are repaired with slow kinetics while the majority is repaired in the first two hours with fast kinetics. γH2AX analysis of ho-mologous recombination (HR) mutants and non-hoho-mologous end-joining (NHEJ) mu-tants show that the DSB repair occurs with the slow and fast kinetics by NHEJ in G1 phase. In G2 phase the majority of DSBs is rejoined with a fast kinetic by NHEJ while the slow kinetic represents HR. Furthermore it could be shown that Artemis and ATM are involved in the slow component in G1 as well as in G2. Since γH2AX foci loss monitors all repair events, alternative techniques which more specifically measure HR of IR-induced DSBs are developed. These techniques show that Artemis and ATM are involved in HR in G2 whereas in G1 Artemis and ATM promote end-joining.
The need of Artemis and ATM in G2 is a consequence of the CtIP-mediated resec-tion of the DSBs. Surprisingly the analysis in G1 show, that DSB end resecresec-tion gene-rates the requirement for Artemis and ATM in the DSB repair in G1 as well as in G2,
probably due to the formation of secondary structures arising during the resection of DSBs. These structures may hinder the proper repair. Because of the well-known endonuclease activity of Artemis it is possible, that Artemis processes these second-ary structures.
Further insights of the function of ATM during HR are provided by the fact that HR repairs preferentially DSBs which are localized in heterochromatic regions. Further-more DSBs repaired with slow kinetics in G1 are also localized in heterochromatic regions. It is known that ATM phosphorylates the heterochromatic building factor Kap1. This phosphorylation contributes to the rearrangement of the Kap1 dependent heterochromatic status. So it can be postulated that ATM is involved in the chromatin remodeling, which is essential for a successful repair of IR induced DSBs. All DSBs which are located in heterochromatic regions are repaired in an ATM dependent manner. Furthermore especially the resected DSBs need the ATM dependent change of heterochromatic status.
This work allows insights into the complex network of DSB repair in G1 and G2. Tak-en together the results show that DSBs repaired by slow kinetics require ATM and Artemis. Strikingly all of these DSBs are resected in G1 and G2 and subcequently repaired by NHEJ and HR. The heterochromatin status or the resection capability can influence the pathway choice. These new insights aim to provide a better under-standing about the repair process of IR-induced DSBs and establish new starting points for prospective works.