Mechanismen der nicht homologen Endverknüpfung in Zellen der höheren Eukaryonten: Funktionen der DNA Ligase III und Substypen von Histone H1
Final Report Abstract
In higher eukaryotes DNA double strand breaks (DSBs) are repaired by homologous recombination (HRR) or non-homologous end joining (NHEJ). Work carried out as part of the present proposal demonstrated that in addition to the DNA-PK dependent pathway of NHEJ (D-NHEJ), cells employ a backup pathway (B-NHEJ) utilizing DNA Ligase III, PARP-1 and possibly also histone H1. Further studies in this project with cells defective in D-NHEJ or HRR and sorted either in Gl or in G2 phase demonstrated enhanced repair of DSBs by B-NHEJ in G2. This result was confirmed with plasmid in vivo and in vitro end joining assays. Furthermore, additional experiments indicated that while D-NHEJ shows no dependence on growth state, the ability of cells to repair DSBs via B-NHEJ is strongly reduced under conditions suppressing cell proliferation. Since elutriated G1 cells from exponentially growing or plateau phase cultures show a response similar to non-elutriated cells, the effect cannot be attributed to redistribution in the cell cycle. An in vitro assay gauging the activity of B-NHEJ in different stages of growth, shows a reduction in DNA end joining during the plateau phase that is corrected by recombinant DNA Ligase IIIa. The results point to interesting cell cycle and cell growth state dependencies of B-NHEJ that generate a framework for investigating the mechanistic basis and the contributions of HRR and D-NHEJ to DSB repair.
Publications
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Cell Line Specific Modulation of Connexin43 Expression after Exposure to Ionizing Radiation. Cell Communication and Adhesion 2005, 12:249-259
Banaz-Yasar, F, Tischka R, Iliakis G, Winterhager E, Gellhaus A
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DNA Ligase III as a Candidate Component of Backup Pathways of Nonhomologous End Joining. Cancer Res. 2005, 65: 4020-4030
Wang H, Rosidi B, Perrault R, Wang M, Zhang L, Windhofer F, Iliakis G
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Postreplicative joining of DNA double-strand breaks causes genomic instability in DNA-PKcs-deficient mouse embryonic fibroblasts. Cancer Res. 2005, 65: 10223-10232
Martin M, Genesca A, Latre L, Jaco I, Taccioli GE, Egozcue J, Blasco MA, Iliakis G, Tusell L
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Computational Methods for Analysis of Foci: Validation for Radiation- Induced y-H2AX Foci in Human Cells. Radiat Res 2006, 165: 113-124
Böcker W, Iliakis G
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PARP-1 and Ku compete for repair of DNA double strand breaks by distinct NHEJ pathways. Nucleic Acids Res. 2006, 34: 6170-6182
Wang M, Wu W, Wu W, Rosidi R, Zhang L, Wang H, Iliakis G
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Backup Pathways of Nonhomologous End Joining May Have a Dominant Role in the Formation of Chromosome Aberrations. In: Chromosomal Alterations, G. Obe, Vijayalaxmi (Eds.), Springer-Verlag Berlin, Heidelberg, New York 2007, 67-85
Iliakis G, Wu W, Wang M, Terzoudi GI, Pantelias GE
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Homology-Directed Repair is Required for the Development of Radioresistance during S-Phase: Interplay between Double-Strand Break Repair and Checkpoint Response. Radiat Res 2007, 167: 1-11
Tamulevicius P, Wang M, Iliakis G
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Low levels of DNA ligases III and IV sufficient for effective NHEJ. J Cell Physiol 2007, 213: 475-483
Windhofer F, Wu W, Iliakis G
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Marked dependence on growth state of backup pathways of NHEJ. Int J Radiat Oncol Biol Phys, 2007, 68: 1462-1470
Windhofer F, Wu W, Wang M, Sing SK, Saha J, Rosidi B, Iliakis G
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DNA double strand break repair inhibition as a cause of heat radiosensitization: Re-evaluation considering backup pathways of NHEJ. Int J Hyperthermia 2008, 24:17-29
Iliakis G, Wu W, Wang M
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Repair of radiation induced DNA double strand breaks by backup NHEJ is enhanced in G2. DNA Repair 2008, 7: 329-338
Wu W, Wang M, Wu W, Singh SK, Mußfeldt T, Iliakis G
