Contribution of the homologous recombination pathway in DNA double-strand break repair of the haploid model plant Physcomitrella patens

[Introduction] Although many plants show radioresistance, the mechanism has not been fully elucidated. Physcomitrella patens is a moss plant that has a genome size of 511 Mb and 17 chromosomes and spends most of its life cycle in haploidy. The purpose of this study is to elucidate the mechanism of radioresistance found in P. patens. [Methods] Knockout strains of RAD51B, LIG4 and POLQ genes, which are important in homologous recombination, non-homologous end joining and alternative end joining repair, were kindly gifted from Dr. Nogué of INRA. Single cells were obtained by treating plant tissues with cell wall degrading enzymes. Plant tissues and single cells were irradiated with 60Co gamma rays and 18.3 MeV/u carbon ions (LET = 108 keV/μm). To measure the radiosensitivity, the irradiated plant tissues were grown for 6 days and then the dry weight was measured. The irradiated single cells were cultured for 2 weeks, and then colony forming ability was measured. To measure the radiation-induced DNA damage and its repair rate, DNA double strand breaks (DSB) were detected by pulsed field gel electrophoresis. [Results] Compared to the wild strain, the lig4 strain was slightly more radiosensitive. The polq strain was slightly more resistant at low dose, but was more sensitive at high dose. The radiosensitivity was significantly enhanced in the rad51b strain. The repair rate of DSB in the wild strain was slower compared to angiosperm tobacco and mammalian cells. [Conclusion] P. patens repairs most DSBs via the homologous recombination pathway.日本放射線影響学会第62回大

Distinct modes of death in human neural stem and glioblastoma cells irradiated with carbon-ion radiation and gamma-rays

Purpose: Accumulated damage in neural stem cells (NSCs) during brain tumor radiotherapy causes cognitive dysfunction to the patients. Carbon-ion radiotherapy can reduce undesired irradiation of normal tissues more efficiently than conventional photon radiotherapy. This study elucidates the responses of NSCs to carbon-ion radiation. Methods: Human NSCs and glioblastoma A-172 cells were irradiated with carbon-ion radiation and gamma-rays, which have different linear-energy-transfer (LET) values of 108 and 0.2 keV/μm, respectively. After irradiation, growth rates were measured, apoptotic cells were detected by flow cytometry, and DNA synthesizing cells were immunocytochemically visualized. Results: Growth rates of NSCs and A-172 cells were decreased after irradiation. The percentages of apoptotic cells were remarkably increased in NSCs but not in A-172 cells. In contrast, the fractions of DNA synthesizing A-172 cells were decreased in a dose-dependent manner. These results indicate that apoptosis induction and DNA synthesis inhibition contribute to the growth inhibition of NSCs and glioblastoma cells, respectively. In addition, high-LET carbon ions induced more profound effects than low-LET gamma-rays. Conclusions: Apoptosis is an important clinical target to protect NSCs during brain tumor radiotherapy using carbon-ion radiation as well as conventional X-rays

相同組換え経路の破綻がヒメツリガネゴケの放射線抵抗性を喪失させる

DNA double-strand breaks (DSBs) are one of the most severe types of DNA damage induced by ionizing radiation (IR) and other causes. We have previously found that the dose of radiation required for cell death in moss Physcomitrella patens is 100-times higher than that in mammals. We also found that, as part of its mechanism, the number of DSBs required for cell death in P. patens is 10-times more than that in mammals. This suggests that P. patens has a superior DSB repair mechanism to mammals. To elucidate the mechanism, we knockout the PpRAD51B, PpLIG4 and PpPOLQ genes, which are key genes in the homologous recombination (HR), non-homologous end joining (NHEJ) and alternative end joining (a-EJ) pathways, respectively, by the CRISPR-Cas9 or the classical recombination method. In survival tests after IR, rad51b strain was 40-times more radiosensitive than other knockout and wildtype strains. This suggests that the HR pathway but not the NHEJ and a-EJ pathways is important for the radioresistance of P. patens. Next, we measured the DSB rejoining after IR by pulsed-field gel electrophoresis. Compared with the wild-type cells, DSB rejoining was delayed in all of rad51b, lig4 and polq cells. This indicates that NHEJ and a-EJ as well as HR are involved in DSB repair. So why was the radiosensitivity significantly increased only in rad51b strain? In the wild-type, lig4 and polq cells, DSB rejoining was completed within 8 to 24 hours after IR. However, in rad51b cells, a part of the DSBs remained un-rejoined even after 24 hours. Taking together, it is suggested that failed NHEJ or a-EJ are easily bucked up by other intact pathways, but failed HR is not.第４３回日本分子生物学会年

Preliminery examination of soft agar colony formation assay for PC12 pheochromocytoma cells

We reported strong anti-tumor effects of α-emitting meta-211At-astato-benzylguanidine (211At-MABG) in a PCC mouse model, suggesting a potential option for targeted α therapy (TAT) for patients with malignant PCC. We also found that the gene expression profiles of cell cycle checkpoints displayed similar modes of cell death via the p53-p21 signaling pathway after 211At-MABG treatment and -ray irradiation. This p53-dependent pathway would induce reproductive cell death, e.g. cell cycle arrest. However, we have not yet checked for reproductive cell death in PCC (PC12) cells by colony formation assays because PC12 cells showed strong aggregation. Here, we preliminary reported the surviving fraction of -ray irradiated PC12 cells using the soft-agar colony formation assay

SOG1 homologues regulate DNA-damage responses in Physcomitrella patens.

A NAC-type transcription factor SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1), found in seed plants, is a master regulator for DNA damage responses (DDR). Upon DNA damage, SOG1 is phosphorylated by the ATM, a conserved Ser/Thr kinase in both animal and plant kingdoms. Then it regulates the expression of a large number of downstream DDR genes. To know the origin of DDR network in land plants, we searched for a homologue(s) of SOG1 in a moss Physcomitrella patens and identified PpSOG1a and PpSOG1b. To assess if either of them, or both, function(s) in DDR, we established knockout plant lines via CRISPR/Cas9 gene editing. Although neither sog1a nor sog1b single mutant showed obviously notable phenotypes, the double knockout sog1a sog1b plants showed reduced expression of DNA-repair genes after gamma-irradiation. The double mutant also showed resistance to several DNA-damaging treatments as similarly seen in Arabidopsis sog1 plants. These results suggest that PpSOG1a and PpSOG1b work redundantly on DDR response in P. patens, and also that the plant specific DDR system had been established prior to the emerging of vascular plants.第62回日本植物生理学会年

SOG1, a plant-specific master regulator of DNA damage responses, originated from nonvascular land plants

The suppressor of gamma response 1 (SOG1), a NAM, ATAF1, 2, and CUC2 (NAC)-type transcription factor found in seed plants, is a master regulator of DNA damage responses (DDRs). Upon DNA damage, SOG1 regulates the expression of downstream DDR genes. To know the origin of the DDR network in land plants, we searched for a homolog(s) of SOG1 in a moss Physcomitrium (Physcomitrella) patens and identified PpSOG1a and PpSOG1b. To assess if either or both of them function(s) in DDR, we knocked out the PpSOG1s using CRISPR/Cas9-mediated gene editing and analyzed the responses to DNA-damaging treatments. The double-knockout (KO) sog1a sog1b plants showed resistance to γ-rays, bleomycin, and ultraviolet B (UVB) treatments similarly seen in Arabidopsis sog1 plants. Next, we irradiated wild-type (WT) and KO plants with γ-rays and analyzed the whole transcriptome to examine the effect on the expression of DDR genes. The results revealed that many P. patens genes involved in the checkpoint, DNA repair, replication, and cell cycle-related genes were upregulated after γ-irradiation, which was not seen in sog1a sog1b plants. These results suggest that PpSOG1a and PpSOG1b work redundantly on DDR response in P. patens; in addition, plant-specific DDR systems had been established before the emergence of vascular plants