ISRIB Blunts the Integrated Stress Response by Allosterically Antagonising the Inhibitory Effect of Phosphorylated eIF2 on eIF2B.

The small molecule ISRIB antagonizes the activation of the integrated stress response (ISR) by phosphorylated translation initiation factor 2, eIF2(αP). ISRIB and eIF2(αP) bind distinct sites in their common target, eIF2B, a guanine nucleotide exchange factor for eIF2. We have found that ISRIB-mediated acceleration of eIF2B's nucleotide exchange activity in vitro is observed preferentially in the presence of eIF2(αP) and is attenuated by mutations that desensitize eIF2B to the inhibitory effect of eIF2(αP). ISRIB's efficacy as an ISR inhibitor in cells also depends on presence of eIF2(αP). Cryoelectron microscopy (cryo-EM) showed that engagement of both eIF2B regulatory sites by two eIF2(αP) molecules remodels both the ISRIB-binding pocket and the pockets that would engage eIF2α during active nucleotide exchange, thereby discouraging both binding events. In vitro, eIF2(αP) and ISRIB reciprocally opposed each other's binding to eIF2B. These findings point to antagonistic allostery in ISRIB action on eIF2B, culminating in inhibition of the ISR.

HCV IRES Captures an Actively Translating 80S Ribosome.

Translation initiation of hepatitis C virus (HCV) genomic RNA is induced by an internal ribosome entry site (IRES). Our cryoelectron microscopy (cryo-EM) analysis revealed that the HCV IRES binds to the solvent side of the 40S platform of the cap-dependently translating 80S ribosome. Furthermore, we obtained the cryo-EM structures of the HCV IRES capturing the 40S subunit of the IRES-dependently translating 80S ribosome. In the elucidated structures, the HCV IRES "body," consisting of domain III except for subdomain IIIb, binds to the 40S subunit, while the "long arm," consisting of domain II, remains flexible and does not impede the ongoing translation. Biochemical experiments revealed that the cap-dependently translating ribosome becomes a better substrate for the HCV IRES than the free ribosome. Therefore, the HCV IRES is likely to efficiently induce the translation initiation of its downstream mRNA with the captured translating ribosome as soon as the ongoing translation terminates.

The Translation Inhibitor Rocaglamide Targets a Bimolecular Cavity between eIF4A and Polypurine RNA.

A class of translation inhibitors, exemplified by the natural product rocaglamide A (RocA), isolated from Aglaia genus plants, exhibits antitumor activity by clamping eukaryotic translation initiation factor 4A (eIF4A) onto polypurine sequences in mRNAs. This unusual inhibitory mechanism raises the question of how the drug imposes sequence selectivity onto a general translation factor. Here, we determined the crystal structure of the human eIF4A1⋅ATP analog⋅RocA⋅polypurine RNA complex. RocA targets the "bi-molecular cavity" formed characteristically by eIF4A1 and a sharply bent pair of consecutive purines in the RNA. Natural amino acid substitutions found in Aglaia eIF4As changed the cavity shape, leading to RocA resistance. This study provides an example of an RNA-sequence-selective interfacial inhibitor fitting into the space shaped cooperatively by protein and RNA with specific sequences.

The Ets transcription factor Spi-B is essential for the differentiation of intestinal microfold cells.

Intestinal microfold cells (M cells) are an enigmatic lineage of intestinal epithelial cells that initiate mucosal immune responses through the uptake and transcytosis of luminal antigens. The mechanisms of M-cell differentiation are poorly understood, as the rarity of these cells has hampered analysis. Exogenous administration of the cytokine RANKL can synchronously activate M-cell differentiation in mice. Here we show the Ets transcription factor Spi-B was induced early during M-cell differentiation. Absence of Spi-B silenced the expression of various M-cell markers and prevented the differentiation of M cells in mice. The activation of T cells via an oral route was substantially impaired in the intestine of Spi-B-deficient (Spib(-/-)) mice. Our study demonstrates that commitment to the intestinal M-cell lineage requires Spi-B as a candidate master regulator.

Autophagy-deficient mice develop multiple liver tumors.

Autophagy is a major pathway for degradation of cytoplasmic proteins and organelles, and has been implicated in tumor suppression. Here, we report that mice with systemic mosaic deletion of Atg5 and liver-specific Atg7⁻/⁻ mice develop benign liver adenomas. These tumor cells originate autophagy-deficient hepatocytes and show mitochondrial swelling, p62 accumulation, and oxidative stress and genomic damage responses. The size of the Atg7⁻/⁻ liver tumors is reduced by simultaneous deletion of p62. These results suggest that autophagy is important for the suppression of spontaneous tumorigenesis through a cell-intrinsic mechanism, particularly in the liver, and that p62 accumulation contributes to tumor progression.

Induction of divalent cation permeability by heterologous expression of a voltage sensor domain.

The voltage sensor domain (VSD) is a protein domain that confers sensitivity to membrane potential in voltage-gated ion channels as well as the voltage-sensing phosphatase. Although VSDs have long been considered to function as regulatory units acting on adjacent effectors, recent studies have revealed the existence of direct ion permeation paths in some mutated VSDs and in the voltage-gated proton channel. In this study, we show that calcium currents are evoked upon membrane hyperpolarization in cells expressing a VSD derived from an ascidian voltage-gated ion channel superfamily. Unlike the previously reported omega-pore in the Shaker K+ channel and rNav1.4, mutations are not required. From electrophysiological experiments in heterologous expression systems, we found that the conductance is directly mediated by the VSD itself and is carried by both monovalent and divalent cations. This is the first report of divalent cation permeation through a VSD-like structure.

Seminal vesicle secretion 2 acts as a protectant of sperm sterols and prevents ectopic sperm capacitation in mice.

Seminal vesicle secretion 2 (SVS2) is a protein secreted by the mouse seminal vesicle. We previously demonstrated that SVS2 regulates fertilization in mice; SVS2 is attached to a ganglioside GM1 on the plasma membrane of the sperm head and inhibits sperm capacitation in in vitro fertilization as a decapacitation factor. Furthermore, male mice lacking SVS2 display prominently reduced fertility in vivo, which indicates that SVS2 protects spermatozoa from some spermicidal attack in the uterus. In this study, we tried to investigate the mechanisms by which SVS2 controls in vivo sperm capacitation. SVS2-deficient males that mated with wild-type partners resulted in decreased cholesterol levels on ejaculated sperm in the uterine cavity. SVS2 prevented cholesterol efflux from the sperm plasma membrane and incorporated liberated cholesterol in the sperm plasma membrane, thereby reversibly preventing the induction of sperm capacitation by bovine serum albumin and methyl-beta-cyclodextrin in vitro. SVS2 enters the uterus and the uterotubal junction, arresting sperm capacitation in this area. Therefore, our results show that SVS2 keeps sterols on the sperm plasma membrane and plays a key role in unlocking sperm capacitation in vivo.

Role of AtPolζ, AtRev1, and AtPolη in UV light-induced mutagenesis in Arabidopsis.

Translesion synthesis (TLS) is a DNA damage tolerance mechanism in which DNA lesions are bypassed by specific polymerases. To investigate the role of TLS activities in ultraviolet light-induced somatic mutations, we analyzed Arabidopsis (Arabidopsis thaliana) disruptants of AtREV3, AtREV1, and/or AtPOLH genes that encode TLS-type polymerases. The mutation frequency in rev3-1 or rev1-1 mutants decreased compared with that in the wild type, suggesting that AtPolζ and AtRev1 perform mutagenic bypass events, whereas the mutation frequency in the polh-1 mutant increased, suggesting that AtPolη performs nonmutagenic bypass events with respect to ultraviolet light-induced lesions. The rev3-1 rev1-1 double mutant showed almost the same mutation frequency as the rev1-1 single mutant. The increased mutation frequency found in polh-1 was completely suppressed in the rev3-1 polh-1 double mutant, indicating that AtPolζ is responsible for the increased mutations found in polh-1. In summary, these results suggest that AtPolζ and AtRev1 are involved in the same (error-prone) TLS pathway that is independent from the other (error-free) TLS pathway mediated by AtPolη.

[Nutritional assessment using MUST(Malnutrition Universal Screening Tool) in outpatient chemotherapy].

OBJECTIVE: We set out to see if nutritional assessment(management)using MUST could be useful for patients undergoing outpatient chemotherapy. METHODS: The study sample consisted of 197 patients undergoing outpatient chemotherapy between June 2010 and November 2010. The results of MUST, serum albumin levels, and nutritional intervention were investigated. RESULTS: High- and medium-risk patients requiring nutritional therapy was comprised of 17/78 breast cancer(21. 8%), 16/63 hematologic malignancy(25. 4%), and 26/56 colonic cancer(46. 4%)patients.Moreover, the serum albumin level in high- and medium-risk patients was likely to decrease compared to low-risk patients, suggesting the usefulness of MUST. DISCUSSION AND CONCLUSION: It is important to assess nutritional status focusedon simplicity, objectivity, andspeedin outpatient chemotherapy. Assessment of patients' nutritional status and cancer treatment compliance are expected to be improved using MUST.

CAMSAP2 organizes a γ-tubulin-independent microtubule nucleation centre through phase separation.

Microtubules are dynamic polymers consisting of αß-tubulin heterodimers. The initial polymerization process, called microtubule nucleation, occurs spontaneously via αß-tubulin. Since a large energy barrier prevents microtubule nucleation in cells, the γ-tubulin ring complex is recruited to the centrosome to overcome the nucleation barrier. However, a considerable number of microtubules can polymerize independently of the centrosome in various cell types. Here, we present evidence that the minus-end-binding calmodulin-regulated spectrin-associated protein 2 (CAMSAP2) serves as a strong nucleator for microtubule formation by significantly reducing the nucleation barrier. CAMSAP2 co-condensates with αß-tubulin via a phase separation process, producing plenty of nucleation intermediates. Microtubules then radiate from the co-condensates, resulting in aster-like structure formation. CAMSAP2 localizes at the co-condensates and decorates the radiating microtubule lattices to some extent. Taken together, these in vitro findings suggest that CAMSAP2 supports microtubule nucleation and growth by organizing a nucleation centre as well as by stabilizing microtubule intermediates and growing microtubules.

Cells are able to hold their shape thanks to tube-like structures called microtubules that are made of hundreds of tubulin proteins. Microtubules are responsible for maintaining the uneven distribution of molecules throughout the cell, a phenomenon known as polarity that allows cells to differentiate into different types with various roles. A protein complex called the γ-tubulin ring complex (γ-TuRC) is necessary for microtubules to form. This protein helps bind the tubulin proteins together and stabilises microtubules. However, recent research has found that in highly polarized cells such as neurons, which have highly specialised regions, microtubules can form without γ-TuRC. Searching for the proteins that could be filling in for γ-TuRC in these cells some evidence has suggested that a group known as CAMSAPs may be involved, but it is not known how. To characterize the role of CAMSAPs, Imasaki, Kikkawa et al. studied how one of these proteins, CAMSAP2, interacts with tubulins. To do this, they reconstituted both CAMSAP2 and tubulins using recombinant biotechnology and mixed them in solution. These experiments showed that CAMSAP2 can help form microtubules by bringing together their constituent proteins so that they can bind to each other more easily. Once microtubules start to form, CAMSAP2 continues to bind to them, stabilizing them and enabling them to grow to full size. These results shed light on how polarity is established in cells such as neurons, muscle cells, and epithelial cells. Additionally, the ability to observe intermediate structures during microtubule formation can provide insights into the processes that these structures are involved in.

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 plant. 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.

eIF2B-capturing viral protein NSs suppresses the integrated stress response.

Various stressors such as viral infection lead to the suppression of cap-dependent translation and the activation of the integrated stress response (ISR), since the stress-induced phosphorylated eukaryotic translation initiation factor 2 [eIF2(αP)] tightly binds to eIF2B to prevent it from exchanging guanine nucleotide molecules on its substrate, unphosphorylated eIF2. Sandfly fever Sicilian virus (SFSV) evades this cap-dependent translation suppression through the interaction between its nonstructural protein NSs and host eIF2B. However, its precise mechanism has remained unclear. Here, our cryo-electron microscopy (cryo-EM) analysis reveals that SFSV NSs binds to the α-subunit of eIF2B in a competitive manner with eIF2(αP). Together with SFSV NSs, eIF2B retains nucleotide exchange activity even in the presence of eIF2(αP), in line with the cryo-EM structures of the eIF2B•SFSV NSs•unphosphorylated eIF2 complex. A genome-wide ribosome profiling analysis clarified that SFSV NSs expressed in cultured human cells attenuates the ISR triggered by thapsigargin, an endoplasmic reticulum stress inducer. Furthermore, SFSV NSs introduced in rat hippocampal neurons and human induced-pluripotent stem (iPS) cell-derived motor neurons exhibits neuroprotective effects against the ISR-inducing stress. Since ISR inhibition is beneficial in various neurological disease models, SFSV NSs may be a promising therapeutic ISR inhibitor.

A UVB-hypersensitive mutant in Arabidopsis thaliana is defective in the DNA damage response.

To investigate UVB DNA damage response in higher plants, we used a genetic screen to isolate Arabidopsis thaliana mutants that are hypersensitive to UVB irradiation, and isolated a UVB-sensitive mutant, termed suv2 (for sensitive to UV 2) that also displayed hypersensitivity to gamma-radiation and hydroxyurea. This phenotype is reminiscent of the Arabidopsis DNA damage-response mutant atr. The suv2 mutation was mapped to the bottom of chromosome 5, and contains an insertion in an unknown gene annotated as MRA19.1. RT-PCR analysis with specific primers to MRA19.1 detected a transcript consisting of 12 exons. The transcript is predicted to encode a 646 amino acid protein that contains a coiled-coil domain and two instances of predicted PIKK target sequences within the N-terminal region. Fusion proteins consisting of the predicted MRA19.1 and DNA-binding or activation domain of yeast transcription factor GAL4 interacted with each other in a yeast two-hybrid system, suggesting that the proteins form a homodimer. Expression of CYCB1;1:GUS gene, which encodes a labile cyclin:GUS fusion protein to monitor mitotic activity by GUS activity, was weaker in the suv2 plant after gamma-irradiation than in the wild-type plants and was similar to that in the atr plants, suggesting that the suv2 mutant is defective in cell-cycle arrest in response to DNA damage. Overall, these results suggest that the gene disrupted in the suv2 mutant encodes an Arabidopsis homologue of the ATR-interacting protein ATRIP.

Structural basis for the exclusive specificity of Slac2-a/melanophilin for the Rab27 GTPases.

Rab27A is required for actin-based melanosome transport in mammalian skin melanocytes through its interaction with a specific effector, Slac2-a/melanophilin. Mutations that disrupt the Rab27A/Slac2-a interaction cause human Griscelli syndrome. The other Rab27 isoform, Rab27B, also binds all of the known effectors of Rab27A. In this study, we determined the crystal structure of the constitutively active form of Rab27B complexed with GTP and the effector domain of Slac2-a. The Rab27B/Slac2-a complex exhibits several intermolecular hydrogen bonds that were not observed in the previously reported Rab3A/rabphilin complex. A Rab27A mutation that disrupts one of the specific hydrogen bonds with Slac2-a resulted in the dramatic reduction of Slac2-a binding activity. Furthermore, we generated a Rab3A mutant that acquires Slac2-a binding ability by transplanting four Rab27-specific residues into Rab3A. These findings provide the structural basis for the exclusive association of Slac2-a with the Rab27 subfamily, whereas rabphilin binds several subfamilies, including Rab3 and Rab27.

Arabidopsis thaliana Y-family DNA polymerase eta catalyses translesion synthesis and interacts functionally with PCNA2.

SUMMARY: Upon blockage of chromosomal replication by DNA lesions, Y-family polymerases interact with monoubiquitylated proliferating cell nuclear antigen (PCNA) to catalyse translesion synthesis (TLS) and restore replication fork progression. Here, we assessed the roles of Arabidopsis thaliana POLH, which encodes a homologue of Y-family polymerase eta (Poleta), PCNA1 and PCNA2 in TLS-mediated UV resistance. A T-DNA insertion in POLH sensitized the growth of roots and whole plants to UV radiation, indicating that AtPoleta contributes to UV resistance. POLH alone did not complement the UV sensitivity conferred by deletion of yeast RAD30, which encodes Poleta, although AtPoleta exhibited cyclobutane dimer bypass activity in vitro, and interacted with yeast PCNA, as well as with Arabidopsis PCNA1 and PCNA2. Co-expression of POLH and PCNA2, but not PCNA1, restored normal UV resistance and mutation kinetics in the rad30 mutant. A single residue difference at site 201, which lies adjacent to the residue (lysine 164) ubiquitylated in PCNA, appeared responsible for the inability of PCNA1 to function with AtPoleta in UV-treated yeast. PCNA-interacting protein boxes and an ubiquitin-binding motif in AtPoleta were found to be required for the restoration of UV resistance in the rad30 mutant by POLH and PCNA2. These observations indicate that AtPoleta can catalyse TLS past UV-induced DNA damage, and links the biological activity of AtPoleta in UV-irradiated cells to PCNA2 and PCNA- and ubiquitin-binding motifs in AtPoleta.

Translesion Synthesis in Plants: Ultraviolet Resistance and Beyond.

Plant genomes sustain various forms of DNA damage that stall replication forks. Translesion synthesis (TLS) is one of the pathways to overcome stalled replication in which specific polymerases (TLS polymerase) perform bypass synthesis across DNA damage. This article gives a brief overview of plant TLS polymerases. In Arabidopsis, DNA polymerase (Pol) ζ, η, κ, θ, and λ and Reversionless1 (Rev1) are shown to be involved in the TLS. For example, AtPolη bypasses ultraviolet (UV)-induced cyclobutane pyrimidine dimers in vitro. Disruption of AtPolζ or AtPolη increases root stem cell death after UV irradiation. These results suggest that AtPolζ and ATPolη bypass UV-induced damage, prevent replication arrest, and allow damaged cells to survive and grow. In general, TLS polymerases have low fidelity and often induce mutations. Accordingly, disruption of AtPolζ or AtRev1 reduces somatic mutation frequency, whereas disruption of AtPolη elevates it, suggesting that plants have both mutagenic and less mutagenic TLS activities. The stalled replication fork can be resolved by a strand switch pathway involving a DNA helicase Rad5. Disruption of both AtPolζ and AtRAD5a shows synergistic or additive effects in the sensitivity to DNA-damaging agents. Moreover, AtPolζ or AtRev1 disruption elevates homologous recombination frequencies in somatic tissues. These results suggest that the Rad5-dependent pathway and TLS are parallel. Plants grown in the presence of heat shock protein 90 (HSP90) inhibitor showed lower mutation frequencies, suggesting that HSP90 regulates mutagenic TLS in plants. Hypersensitivities of TLS-deficient plants to γ-ray and/or crosslink damage suggest that plant TLS polymerases have multiple roles, as reported in other organisms.

Anaphylaxis to Carboxymethylcellulose: Add Food Additives to the List of Elicitors.

A 14-year-old girl developed 4 episodes of anaphylaxis of unknown etiology, which required intramuscular adrenaline administration each time. She had eaten pizza and a cheeseburger immediately before the first 2 episodes, respectively, but had not eaten anything for several hours before the last 2 episodes. It turned out that she had eaten the same ice lolly 4 hours before the first 3 episodes and a Café au lait Swirkle (a half-frozen beverage) 4 hours before the last episode. We detected carboxymethylcellulose sodium as the only common ingredient in all anaphylactic episodes. Skin prick tests were positive for carboxymethylcellulose solution and carboxymethylcellulose-containing food products. We obtained a custom-made carboxymethylcellulose sodium-free ice lolly from the manufacturer and confirmed that it did not induce anaphylactic reactions by a challenge test. Carboxymethylcellulose, an anionic water-soluble polymer derived from native cellulose, is considered to be unabsorbable from the human gut and has been widely and increasingly used in pharmaceutical preparations, cosmetics, and food. This article is the first report of anaphylaxis caused by carboxymethylcellulose-containing foods, whereas anaphylaxis to carboxymethylcellulose has been rarely associated with carboxymethylcellulose-containing pharmaceuticals. Although the exact mechanisms underlying the induction of late-onset anaphylaxis by carboxymethylcellulose remain unclear, a small minority of cellulose-digesting microbial flora in the human colon and contamination of food products with carboxymethylcellulose of low molecular weight might be involved. The induction of recurrent anaphylaxis by various products should be a clue that prompts physicians to suspect food additives as a cause for anaphylaxis.

Structural basis for eIF2B inhibition in integrated stress response.

A core event in the integrated stress response, an adaptive pathway common to all eukaryotic cells in response to various stress stimuli, is the phosphorylation of eukaryotic translation initiation factor 2 (eIF2). Normally, unphosphorylated eIF2 transfers the methionylated initiator tRNA to the ribosome in a guanosine 5'-triphosphate-dependent manner. By contrast, phosphorylated eIF2 inhibits its specific guanine nucleotide exchange factor, eIF2B. To elucidate how the eIF2 phosphorylation status regulates the eIF2B activity, we determined cryo-electron microscopic and crystallographic structures of eIF2B in complex with unphosphorylated or phosphorylated eIF2. The unphosphorylated and phosphorylated forms of eIF2 bind to eIF2B in completely different manners: the nucleotide exchange-active and -inactive modes, respectively. These structures explain how phosphorylated eIF2 dominantly inhibits the nucleotide exchange activity of eIF2B.

Mutator alleles of yeast DNA polymerase zeta.

The yeast REV3 gene encodes the catalytic subunit of DNA polymerase zeta (pol zeta), a B family polymerase that performs mutagenic DNA synthesis in cells. To probe pol zeta mutagenic functions, we generated six mutator alleles of REV3 with amino acid replacements for Leu979, a highly conserved residue inferred to be at the pol zeta active site. Replacing Leu979 with Gly, Val, Asn, Lys, Met or Phe resulted in yeast strains with elevated UV-induced mutant frequencies. While four of these strains had reduced survival following UV irradiation, the rev3-L979F and rev3-L979M strains had normal survival, suggesting retention of pol zeta catalytic activity. UV mutagenesis in the rev3-L979F background was increased when photoproduct bypass by pol eta was eliminated by deletion of RAD30. The rev3-L979F mutation had little to no effect on mutagenesis in an ogg1Delta background, which cannot repair 8-oxo-guanine in DNA. UV-induced can1 mutants from rev3-L979F and rad30Deltarev3-L979F strains primarily contained base substitutions and complex mutations, suggesting error-prone bypass of UV photoproducts by L979F pol zeta. Spontaneous mutation rates in rev3-L979F and rev3-L979M strains are elevated by about two-fold overall and by two- to eight-fold for C to G transversions and complex mutations, both of which are known to be generated by wild-type pol zetain vitro. These results indicate that Rev3p-Leu979 replacements reduce the fidelity of DNA synthesis by yeast pol zetain vivo. In conjunction with earlier studies, the data establish that the conserved amino acid at the active site location occupied by Leu979 is critical for the fidelity of all four yeast B family polymerases. Reduced fidelity with retention of robust polymerase activity suggests that the homologous rev3-L979F allele may be useful for analyzing pol zeta functions in mammals, where REV3 deletion is lethal.

The Moss Physcomitrella patens Is Hyperresistant to DNA Double-Strand Breaks Induced by γ-Irradiation.

The purpose of this study was to investigate whether the moss Physcomitrella patens cells are more resistant to ionizing radiation than animal cells. Protoplasts derived from P. patens protonemata were irradiated with γ-rays of 50-1000 gray (Gy). Clonogenicity of the protoplasts decreased in a γ-ray dose-dependent manner. The dose that decreased clonogenicity by half (LD50) was 277 Gy, which indicated that the moss protoplasts were 200-times more radioresistant than human cells. To investigate the mechanism of radioresistance in P. patens, we irradiated protoplasts on ice and initial double-strand break (DSB) yields were measured using the pulsed-field gel electrophoresis assay. Induced DSBs linearly increased dependent on the γ-ray dose and the DSB yield per Gb DNA per Gy was 2.2. The DSB yield in P. patens was half to one-third of those reported in mammals and yeasts, indicating that DSBs are difficult to induce in P. patens. The DSB yield per cell per LD50 dose in P. patens was 311, which is three- to six-times higher than those in mammals and yeasts, implying that P. patens is hyperresistant to DSBs. Physcomitrella patens is indicated to possess unique mechanisms to inhibit DSB induction and provide resistance to high numbers of DSBs.