The Ubiquitin Ligase RNF138 Cooperates with CtIP to Stimulate Resection of Complex DNA Double-Strand Breaks in Human G1-Phase Cells

DNA double-strand breaks (DSBs) represent the molecular origin of ionizing-radiation inflicted biological effects. An increase in the ionization density causes more complex, clustered DSBs that can be processed by resection also in G1 phase, where repair of resected DSBs is considered erroneous and may contribute to the increased biological effectiveness of heavy ions in radiotherapy. To investigate the resection regulation of complex DSBs, we exposed G1 cells depleted for different candidate factors to heavy ions or α-particle radiation. Immunofluorescence microscopy was used to monitor the resection marker RPA, the DSB marker γH2AX and the cell-cycle markers CENP-F and geminin. The Fucci system allowed to select G1 cells, cell survival was measured by clonogenic assay. We show that in G1 phase the ubiquitin ligase RNF138 functions in resection regulation. RNF138 ubiquitinates the resection factor CtIP in a radiation-dependent manner to allow its DSB recruitment in G1 cells. At complex DSBs, RNF138′s participation becomes more relevant, consistent with the observation that also resection is more frequent at these DSBs. Furthermore, deficiency of RNF138 affects both DSB repair and cell survival upon induction of complex DSBs. We conclude that RNF138 is a regulator of resection that is influenced by DSB complexity and can affect the quality of DSB repair in G1 cells

Investigations to the DNA resection regulation of ion-induced DSBs in G1 cells and the resection limitations of the cells in quiescent state

Complex DNA double-strand breaks (DSB) are the most severe DNA damage in that they represent an enormous challenge for the cell to repair faithfully as well as repairing at all. Two main repair pathways are known to repair DSBs: classical non-homologous end joining (c-NHEJ) and homologous recombination (HR). c-NHEJ is available in all cell cycle phases whereas the HR pathway functions only in S/G2 when the sister chromatid is available as a template. In addition, a further repair pathway is available to the cells – alternative non-homologous end joining (alt- NHEJ), which can operate on resected DNA break ends. DNA resection is an important step in the cell’s decision as to which DSB repair pathway to choose. DNA resection takes place not only in S/G2 cells but also in G1 cells following complex DNA damage induced by heavy ion radiation. The goal of this thesis is to elucidate the mechanism of resection regulation in G1 cells after heavy ion irradiation. It specifically focuses on the role of the resection regulatory factors RIF1 and BRCA1. A useful model to study the mechanism of resection limitation are cells in quiescent state, G0 cells. In this thesis, quiescent human fibroblasts were used due to their limited resection of heavy ion- induced DSBs in comparison to proliferating human fibroblasts in G1 phase. Therefore, the further aim was to investigate the role of quiescent state compared to the proliferating state in the resection regulation after low- and high LET irradiation. Recruitment of RIF1 to heavy ion induced DSBs in S/G2 and G1 phase cells suggested its involvement in the DNA damage response of complex DNA lesions. RIF1’s recruitment to DSB sites is LET dependent in S/G2 cells. However, a direct impairment of DNA resection of ion- induced DSBs by RIF1 was not detected. An RPA-foci formation analysis upon ion irradiation in BRCA1 depleted cells, however, it showed that the RIF1 antagonist BRCA1 is involved in resection regulation of ion-induced DSBs in G1 cells. Moreover, BRCA1 depletion resulted in a strong increase of the number of RIF1 foci in G1 cells after carbon ion irradiation suggesting that RIF1 is, after all, involved in the resection regulation of heavy ion induced DSBs. This was further supported by the observation that RIF1 depletion led to an increase of the BRCA1 foci number in G1 cells. Co-immunofluorescence staining of BRCA1 and RIF1 showed cell cycle dependent overlap of the BRCA1 and RIF1 foci, which was more pronounced in G1 than in S/G2 cells. The observed overlap of BRCA1 and RPA foci was also cell cycle dependent, yet was greater in S/G2 compared to G1 cells suggesting that BRCA1 is more active in the resection promotion after heavy ion radiation in S/G2 than in G1 cells. UHRF1 is as an interaction partner of BRCA1 in removing RIF1 from DSBs. Therefore, UHRF1 was considered as a positive resection regulator and hence was depleted in U2OS cells to analyze its influence on resection of ion-induced DSBs. Surprisingly, UHRF1 depletion strongly decreased the fraction of resection positive G1 and S/G2 cells indicating a significant role of UHRF1 in the resection regulation of complex DSBs. Normal human fibroblasts in G0 phase showed a smaller number of resection positive cells than fibroblasts in G1 phase. To analyze the cause of decreased resection in human fibroblasts a XI protocol to enrich the cells in G1 or G0 phase was established. The biochemical characteristics of human fibroblasts in G1 and G0 phase as well as DSB repair-kinetics after X-ray irradiation showed essential differences between these two cell cycle states. The examination of repair factors that promote resection by Western analysis revealed that BRCA1 and CtIP were strongly reduced in quiescent cells. Moreover, the resection antagonists 53BP1 and Ku80, which protect the DSB ends from resection to promote c-NHEJ, were available. Interestingly, RIF1, which represents another resection antagonist, was not detectable in quiescent cells. The RIF1 downregulation is in line with its recently discovered role in the DNA replication machinery. Furthermore, together with the decreased fraction of resection positive cells after heavy ion irradiation, G0 cells showed slower repair kinetics of X-ray induced DSBs than fibroblasts in G1 phase. A 24 h post irradiation G0 cells revealed still 20 - 30 % unrepaired DSBs detected by gH2AX immunofluorescence staining whereas DSBs in G1 cells were virtually all repaired. The possibility that G0 and G1 cells utilize different DSB repair pathways was examined by using DNA-repair pathway inhibitors; a DNA-PKcs inhibitor was employed in order to inhibit c-NHEJ and a PARP inhibitor was used to inhibit alt-NHEJ. The severe repair impairment of X-ray induced DSBs by the DNA-PKcs inhibitor and the lack of influence by the PARP inhibitor indicated that the main repair pathway for both G1 and G0 cells is c-NHEJ to repair this type of damage. Interestingly, the repair kinetics of helium ion-induced DSBs in G1 and G0 cells was identical, suggesting that c-NHEJ represents the main repair pathway. Analysis of heterochromatin marker trimethylated histone H3 at amino acids lysine 9 (H3K9Me3) and lysine 27 (H3K27Me3) suggested that G0 cells have more compact chromatin than G1 cell. This may well be the reason why G0 cells show different repair kinetics of X-ray induced DSBs compared to G1 cells, as the chromatin status influences the DSB-repair pathway choice and thus repair kinetics. The comparative analysis by clonogenic survival assay of delayed plated G1 versus G0 cells after ionizing radiation mirrored the repair kinetics after X-ray- and heavy ion irradiation. Post X-ray irradiation G0 cells were more sensitive than G1 cells, whereas after carbon ion irradiation the G0 and G1 cells showed almost the same radiosensitivity.Taken together this thesis showed, that both G1 and G0 cells can efficiently repair heavy ion induced damage by DNA-PKcs dependent repair pathway. However, a different chromatin structure might be a cause of slower repair kinetics in G0 cells after X-ray irradiation compared to G1 cells. A slower repair kinetics after Helium ion irradiation compared to X-ray irradiation in G1 cells suggests that G1 cells use a resection dependent c-NHEJ to repair complex DSBs

The Ubiquitin Ligase RNF138 Cooperates with CtIP to Stimulate Resection of Complex DNA Double-Strand Breaks in Human G1-Phase Cells

DNA double-strand breaks (DSBs) represent the molecular origin of ionizing-radiation inflicted biological effects. An increase in the ionization density causes more complex, clustered DSBs that can be processed by resection also in G1 phase, where repair of resected DSBs is considered erroneous and may contribute to the increased biological effectiveness of heavy ions in radiotherapy. To investigate the resection regulation of complex DSBs, we exposed G1 cells depleted for different candidate factors to heavy ions or α-particle radiation. Immunofluorescence microscopy was used to monitor the resection marker RPA, the DSB marker γH2AX and the cell-cycle markers CENP-F and geminin. The Fucci system allowed to select G1 cells, cell survival was measured by clonogenic assay. We show that in G1 phase the ubiquitin ligase RNF138 functions in resection regulation. RNF138 ubiquitinates the resection factor CtIP in a radiation-dependent manner to allow its DSB recruitment in G1 cells. At complex DSBs, RNF138′s participation becomes more relevant, consistent with the observation that also resection is more frequent at these DSBs. Furthermore, deficiency of RNF138 affects both DSB repair and cell survival upon induction of complex DSBs. We conclude that RNF138 is a regulator of resection that is influenced by DSB complexity and can affect the quality of DSB repair in G1 cells

The Ubiquitin Ligase RNF138 Cooperates with CtIP to Stimulate Resection of Complex DNA Double-Strand Breaks in Human G1-Phase Cells

DNA double-strand breaks (DSBs) represent the molecular origin of ionizing-radiation inflicted biological effects. An increase in the ionization density causes more complex, clustered DSBs that can be processed by resection also in G1 phase, where repair of resected DSBs is considered erroneous and may contribute to the increased biological effectiveness of heavy ions in radiotherapy. To investigate the resection regulation of complex DSBs, we exposed G1 cells depleted for different candidate factors to heavy ions or α-particle radiation. Immunofluorescence microscopy was used to monitor the resection marker RPA, the DSB marker γH2AX and the cell-cycle markers CENP-F and geminin. The Fucci system allowed to select G1 cells, cell survival was measured by clonogenic assay. We show that in G1 phase the ubiquitin ligase RNF138 functions in resection regulation. RNF138 ubiquitinates the resection factor CtIP in a radiation-dependent manner to allow its DSB recruitment in G1 cells. At complex DSBs, RNF138′s participation becomes more relevant, consistent with the observation that also resection is more frequent at these DSBs. Furthermore, deficiency of RNF138 affects both DSB repair and cell survival upon induction of complex DSBs. We conclude that RNF138 is a regulator of resection that is influenced by DSB complexity and can affect the quality of DSB repair in G1 cells