Radiation-Induced Cell Cycle Checkpoints

Sunday, October 16, 2005 10:15 AM-12:00 PM Room No. 710/712
Chair(s): Syljuåsen, Randi

(SY010) Pathways of DNA double strand break repair and checkpoint response.

Iliakis, George*,1, Wang, Huichen2, Rosidi, Bustanur1, Wang, Minli1, Windhofer, Frank1, Zhang, Lihua1, Wang, Ya, 1 Institute of Medical Radiation Biology, Essen, NRW, Germany2 Department of Radiation Oncology, Philadelphia, PA, USA

ABSTRACT- Biochemical and genetic studies support the view that the majority of DNA double strand breaks (DSB) induced in the genome of higher eukaryotes by ionizing radiation (IR) are removed by two pathways of nonhomologous end joining (NHEJ) termed D-NHEJ and B-NHEJ. Homologous recombination (HR) also contributes to the repair of DSBs as evidenced by the increased radiosensitivity of cells deficient in factors required for this process, but the exact contribution to the removal of DSBs remains unknown. While D-NHEJ depends on the activities of the DNA-dependent protein kinase (DNA-PK) and DNA ligase IV/XRCC4, components of B-NHEJ have only recently been identified. Using extract fractionation we showed that the majority of DNA end joining activity in extracts of HeLa cells derives from DNA ligase III. DNA ligase III fractionates through two columns with the maximum in DNA end joining activity and its depletion from the extract causes loss of activity that can be recovered by the addition of purified enzyme. An in vivo plasmid assay shows that DNA ligase IV deficient mouse embryo fibroblasts retain significant DNA end joining activity that can be reduced by up to 80% by knocking down DNA ligase III using RNAi. Additional experiments implicate PARP-1 in the same backup pathways of NHEJ. These observations identify, in addition to the DNA-PK dependent NHEJ and HR, also B-NHEJ as an essential pathway for the rejoining of radiation induced DNA DSBs and raise the question regarding connection between DSB repair and checkpoint response. Available evidence supports the view that known checkpoints connect mainly to HR. A model for such connection based on published results will be presented and a possible mechanism for checkpoint response during NHEJ outlined. Supported by grants from the Volkswagenstiftung and the DFG, as well as by Contract No FIGH-CT-2002-00217 awarded by the European Commission. Early stages of the work were supported by grants RO1 CA42026, RO1 CA56706 awarded from NIH, DHHS, and a grant from the IFORES program of the University of Duisburg-Essen. H. Wang was supported by T32 CA09137 awarded by NIH, NCI, DHHS.

Key words: Homologous Recombination Repair, DNA Double Strand Breaks, NHEJ, Checkpoints

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2005 RRS