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Editorial: Maintenance of genome integrity; DNA damage sensing, signaling, repair, and replication in plants

Balestrazzi, A.*; Achary V Mohan Murali*; Macovei, A.*; 愿山 郁*; 坂本 綾子

Frontiers in Plant Science (Internet), 7, p.64_1 - 64_2, 2016/02

 被引用回数:3 パーセンタイル:52.83(Plant Sciences)



Evaluation of DNA lesions induced by high-let ion beams using quantitative polymerase chain reaction

松尾 陽一郎*; 泉 佳伸*; 長谷 純宏; 坂本 綾子; 清水 喜久雄*

JAEA-Review 2015-022, JAEA Takasaki Annual Report 2014, P. 104, 2016/02



Knockout of the ${{it OsNBS1}}$ gene in rice via targeted mutagenesis using the CRISPR/Cas9 system

雑賀 啓明*; 三上 雅史*; 遠藤 真咲*; 坂本 綾子; 土岐 精一*

JAEA-Review 2015-022, JAEA Takasaki Annual Report 2014, P. 97, 2016/02

Arabidopsis thaliana cells, in which DNA double-strand breaks (DSBs) have occurred, select either DNA repair, cell death or endocycle. Avoidance of entry into endocycle even under genotoxic conditions, rice (Oryza sativa L.) is more suitable than Arabidopsis for the study of DSBs repair in plants. Nijmegen breakage syndrome 1 (NBS1) protein consists of the Mre11/Rad50/NBS1 (MRN) complex plays an important role in response to DNA damage in mammal. Although rice has a homologue of NBS1, OsNBS1, its function remains unclear. Here, we attempted to knockout the OsNBS1 gene using the recently developed clustered regularly interspaced short palindromic repeat (CRISPR)-associated endonuclease 9 (CRISPR/Cas9) system. Rice (cv. Nipponbare) cells were transformed via Agrobacterium and obtained 72 lines of cali. Cleaved amplified polymorphic sequences (CAPS) analysis detected mutations in 58 lines. Biallelic mutations in theOsNBS1 gene were induced in 24 lines. Sequence analyses of PCR products in #2 and #4 detected not only small mutations but also large modifications at the expected cleavage site. The large insertions involved one or two fragments that seem to be copied from sequences around the cleavage site. These mutations are thought to be repaired by a synthesis-dependent strand annealing (SDSA)-like system as proposed in a previous report in barley.


Molecular analysis of heavy ion induced mutations in budding yeast ${it S. cerevisiae}$

松尾 陽一郎*; 泉 佳伸*; 長谷 純宏; 坂本 綾子; 清水 喜久雄*

JAEA-Review 2014-050, JAEA Takasaki Annual Report 2013, P. 119, 2015/03

高LETのイオンビームによる突然変異誘発の分子機構を明らかにするために、出芽酵母(${it Saccharomyces cerevisiae}$)を材料として研究を行ってきた。これまでに、LETの増加に伴って致死率および${it URA3}$遺伝子の突然変異率が上昇する傾向があること、ならびに突然変異の分布としてヌクレオソーム構造のリンカーDNAにおいて局所的に突然変異が誘発されることを示唆する結果を報告した。ここでは、突然変異が誘発される部位がヌクレオソーム構造に関係があるという仮説を検証するために、野生株と異なるヌクレオソーム構造の${it URA3}$遺伝子を持つ組換え株を作成し、突然変異誘発スペクトルを分析した。野生株での突然変異の位置と、ヌクレオソーム構造が異なる株での${it URA3}$遺伝子の突然変異位置には相違があり、突然変異が生じる位置がヌクレオソーム構造に依存することが示唆された。


Analysis of DNA strand breaks induced by carbon ion beams in ${{it Arabidopsis}}$

寺西 美佳*; 山口 弘子*; 坂本 綾子; 日出間 純*

JAEA-Review 2014-050, JAEA Takasaki Annual Report 2013, P. 115, 2015/03

Ion beams induce DNA damage, such as strand breaks, possibly resulting in an increased mutation frequency. To elucidate the mechanism of mutation induction in plants, it is important to know about which DNA damage converted into a mutation. In this study, we examined the method for determining the number of DNA strand breaks induced by carbon ion beams in Arabidopsis seedlings. Seeds of Arabidopsis thaliana (wild-type Colombia) were aseptically sown on Murashige and Skoog agar plates. The seedlings were irradiated with carbon ion beams from an AVF cyclotron. After irradiation, whole plants were harvested immediately in order to detect the induced DNA strand breaks. The number of DNA strand breaks increased with increasing dose of carbon ion beams. Irradiation of seedlings with 960 Gy of carbon ion beams resulted in up to 2.3 strand breaks per megabase (Mb). In this study, only DNA double strand breaks can be detected using the non-denatured agarose gel electrophoresis. To elucidate the mechanism of mutation induction in plants, measurement of the extent of DNA damage other than DNA strand breaks should be needed. Further analysis is in progress.


Molecular analysis of heavy ion induced mutations in budding yeast ${it S. cerevisiae}$

松尾 陽一郎*; 泉 佳伸*; 長谷 純宏; 坂本 綾子; 野澤 樹; 鳴海 一成*; 清水 喜久雄*

JAEA-Review 2013-059, JAEA Takasaki Annual Report 2012, P. 112, 2014/03

To investigate the nature of mutations induced by accelerated ions in eukaryotic cells, the effects of carbon-ion irradiation were compared with those of $$gamma$$-ray irradiation in the budding yeast ${it Saccharomyces cerevisiae}$. Previous studies suggested that the mutation sites induced by carbon ions were localized near the linker regions of nucleosomes, whereas mutations induced by $$gamma$$ rays were located uniformly throughout the gene. We hypothesized that the locus of mutations might be related to the nucleosome structure. To confirm this hypothesis, we examined the mutation spectrum in the ${it URA3}$ gene with the altered nucleosome structure. It is likely that sites of mutations occurred in the ${it URA3}$ with altered nucleosome structure is inconsistent with those in the wild type. We will further accumulate the data to examine the above hypothesis.


Isolation of a novel UVB-tolerant rice mutant obtained by exposure to carbon-ion beams

高野 成央*; 高橋 祐子*; 山本 充*; 寺西 美佳*; 山口 弘子*; 坂本 綾子; 長谷 純宏; 藤澤 弘子*; Wu, J.*; 松本 隆*; et al.

Journal of Radiation Research, 54(4), p.637 - 648, 2013/07

 被引用回数:11 パーセンタイル:51.49(Biology)

UVB radiation suppresses photosynthesis and protein biosynthesis in plants, which in turn decreases growth and productivity. Here, an ultraviolet-B (UVB)-tolerant rice mutant, ${it utr319}$ (${it UV Tolerant Rice 319}$), was isolated from a mutagenized population derived from 2,500 M1 seeds that were exposed to carbon ions. The ${it utr319}$ mutant was more tolerant to UVB than the wild type. Neither the levels of UVB-induced CPDs, nor the repair of cyclobutane pyrimidine dimers or (6-4) pyrimidine-pyrimidone photodimers was altered in the ${it utr319}$ mutant. Thus, the ${it utr319}$ mutant may be impaired in the production of a previously unidentified factor that confers UVB tolerance. To identify the mutated region in the ${it utr319}$ mutant, microarray-based comparative genomic hybridization analysis was performed. Two adjacent genes on chromosome 7were predicted to represent the mutant allele. Sequence analysis of the chromosome region in ${it utr319}$ revealed a deletion of 45,419 bp. Database analysis indicated that the Os07g0265100 gene, ${it UTR319}$, encodes a putative protein with unknown characteristics or function. In addition, the homologs of UTR319 are conserved only among land plants. Therefore, ${it utr319}$ is a novel UVB-tolerant rice mutant and UTR319 may be crucial for the determination of UVB sensitivity in rice, although the function of UTR319 has not yet been determined.


Mutational effects of $$gamma$$-rays and carbon ion beams in ${it Arabidopsis}$ seedlings

吉原 亮平*; 野澤 樹; 長谷 純宏; 鳴海 一成; 日出間 純*; 坂本 綾子

Journal of Radiation Research, 54(6), p.1050 - 1056, 2013/05

 被引用回数:16 パーセンタイル:62.12(Biology)

To assess the mutational effects of radiations in vigorously proliferating plant tissue, mutation spectrum was analyzed with ${it Arabidopsis}$ seedlings via plasmid rescue method. Transgenic plants containing ${it Escherichia coli rpsL}$ gene were irradiated with $$gamma$$-rays and carbon ion beams (320 MeV $$^{12}$$C$$^{6+}$$), and mutations in the ${it rpsL}$ gene were analyzed. Mutant frequency was significantly increased by $$gamma$$-rays, but not by 320 MeV $$^{12}$$C$$^{6+}$$. Mutation spectrum showed that both radiations increased the frequency of frameshifts and other mutations including deletion/insertion but that only $$gamma$$-rays increased the frequency of total base substitutions. These results suggest that the type of DNA lesions which cause base substitutions were less induced by 320 MeV $$^{12}$$C$$^{6+}$$ than by $$gamma$$-rays in ${it Arabidopsis}$ seedlings. $$gamma$$-rays never increased the frequencies of G:C to T:A and A:T to C:G transversions, which are caused by oxidized guanine, though 320 MeV $$^{12}$$C$$^{6+}$$ slightly increase the both transversions. Instead, $$gamma$$-rays significantly increased the frequency of G:C to A:T transition. These results suggest that 8-oxoguanine has little effect on mutagenesis in ${it Arabidopsis}$ cells.


Effect of carbon ion beam irradiation on callus growth in Arabidopsis and rice

雑賀 啓明*; 吉原 亮平*; 坂本 綾子; 土岐 精一*

JAEA-Review 2012-046, JAEA Takasaki Annual Report 2011, P. 99, 2013/01

To date, many commercial varieties of various kinds of plants have been developed by ion beam breeding. However, to further improve the ion beam breeding system, it is important to gather more basic information on the mutagenic effects of ion beams in plants. To this end, we try to establish a novel mutation detection system using a conditional negative selection marker to stop cell growth. Cultured cell such as callus that can clonally propagate efficiently and easily are well-suited to this system. In this study, we checked the sensitivity of callus of the model plants Arabidopsis and rice to carbon ion beams.


DNA damage evaluation system of the high-LET ion beam using the polymerase chain reaction

松尾 陽一郎*; 泉 佳伸*; 長谷 純宏; 坂本 綾子; 野澤 樹; 鳴海 一成; 清水 喜久雄*

JAEA-Review 2012-046, JAEA Takasaki Annual Report 2011, P. 105, 2013/01

We have been studying ion beam-induced mutations in budding yeast S288c (${it RAD}$ $$^{+}$$) as a model of eukaryote cell. We report a new method to evaluate DNA lesions caused by high-LET radiation using the polymerase chain reaction (PCR). PCR is one of the most reliable methods for detecting DNA damage as the amplification stops at the site of the damage. In this study, the 804-bp region of ${it URA3}$ gene was amplified by PCR reaction using a specific oligonucleotide primer set. The PCR device adopted was an Eco Real-Time PCR System (Illumina). The percentage of undamaged template DNA was tended to decrease with an increase in absorbed dose of radiation. The higher LET radiations resulted in the higher rate of decrease in undamaged template DNA. This result suggests that different types of lesions are produced on DNA depending on the LET value of radiations.


Analysis of mutated region on DNA in ion beam-induced UVB tolerant or sensitive rice mutant

高野 成央*; 高橋 祐子*; 山本 充*; 寺西 美佳*; 長谷 純宏; 坂本 綾子; 田中 淳; 日出間 純*

JAEA-Review 2011-043, JAEA Takasaki Annual Report 2010, P. 104, 2012/01

UVB radiation can affect the plant growth and development. The aim of this study is identify new factor(s) related to UVB tolerance in plants by bioengineering or breeding programs. To produce UVB tolerant or sensitive mutants, dry seeds of Japanese rice cultivar Sasanishiki were exposed with carbon ion beams, and two UVB tolerant and three UVB sensitive mutants were isolated from the offspring of irradiated plants. By analysis of comparative genomic hybridization and sequencing of bacterial artificial chromosome library, we identified the mutated regions in a UVB tolerant mutant ${it utr319}$ and a UVB sensitive mutant ${it usr1}$.


Fundamental study on molecular mechanism underlying repair of heavy-ion induced DNA damage in the ${it Saccharomyces cerevisiae}$

松尾 陽一郎*; 泉 佳伸*; 長谷 純宏; 坂本 綾子; 野澤 樹; 鳴海 一成; 清水 喜久雄*

JAEA-Review 2011-043, JAEA Takasaki Annual Report 2010, P. 108, 2012/01

We have been studying ion-beam induced mutations in the budding yeast as a model of eukaryote cell. Yeast cells were irradiated with 220 MeV carbon ions with 107 keV/$$mu$$m LET. The survival rates following irradiation were determined on the basis of colony-forming ability. ${it rad50}$ and ${it rad52}$ strains showed hyper sensitivity, while the ${it ogg1}$ and ${it msh2}$ strains showed relatively lower sensitivity to the carbon ion irradiation. The expression of ${it RAD50}$ gene was up-regulated following carbon ion irradiation but not $$gamma$$ rays. This difference may result from the repair pathway that operates in mutant strains.


Role of AtPol$$zeta$$, AtRev1 and AtPol$$eta$$ in $$gamma$$-ray-induced mutagenesis

中川 繭*; 高橋 真哉*; 鳴海 一成; 坂本 綾子

Plant Signaling & Behavior (Internet), 6(5), p.728 - 731, 2011/05

Ionizing radiations have been applied to plants as useful mutagens, but its molecular mechanism(s) for mutagenesis is less understood. The AtPol$$zeta$$, AtRev1 and AtPol$$eta$$ are Arabidopsis translesion synthesis (TLS)-type polymerases involved in the UV-induced mutagenesis. To investigate a role(s) of TLS-type DNA polymerases in radiation-induced mutagenesis, we analyzed the mutation frequency in AtPol$$zeta$$-, AtRev1- or AtPol$$eta$$-knockout plants, ${it rev3-1}$, ${it rev1-1}$ and ${it polh-1}$, respectively. The mutation frequency in ${it rev3-1}$ was little changed, whereas the frequency in ${it rev1-1}$ greatly decreased and in ${it polh-1}$ slightly increased compared to that of wild type. Abasic (apurinic/apyrimidinic; AP) site, induced by radiations or generated during DNA repair process, leads to incorporation of any kinds of nucleotides at the opposite strand. The 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG), induced by radiation through the formation of reactive oxygen species, makes pairs with cytosine and adenines. Therefore, AtRev1 possibly inserts dC opposite AP-site or 8-oxo-dG, which results in G to T transversions.


Mutational effect of $$gamma$$-rays and carbon ion beams on ${it Arabidopsis}$ seedlings

吉原 亮平; 長谷 純宏; 野澤 樹; 坂本 綾子; 鳴海 一成

JAEA-Review 2010-065, JAEA Takasaki Annual Report 2009, P. 59, 2011/01

To elucidate the molecular mechanisms of mutagenesis by ion beams in higher plant seedlings, mutational effects of ion beams an $$gamma$$-rays were investigated. Mutant frequency was increased by the irradiation of both radiations. Deletion/insertion and G to A transition were major mutations induced by both radiations. However, base change mutations induced by oxidized guanine (G to T and A to C) were not increased in ${it Arabidopsis}$ seedlings as in dry seeds, suggesting that the effects of oxidized guanine induced by ionizing radiation may be lower in plant than in other organisms irrespective of LET.


Effect of different LET radiations on root growth of ${it Arabidopsis thaliana}$

吉原 亮平; 野澤 樹; 雑賀 啓明*; 寺西 美佳*; 土岐 精一*; 日出間 純*; 坂本 綾子

JAEA-Review 2010-065, JAEA Takasaki Annual Report 2009, P. 74, 2011/01

This study aimed to elucidate the effects of ion beams on ${it Arabidopsis thaliana}$. We focus on (1) what kinds of damage are produced by ion beams, and (2) what kinds of cellular processes are involved in turn the damage into mutations. Our results suggest that 220 MeV carbon ions may induce more numbers of DNA lesions or more severe types of DNA lesions than $$gamma$$-rays and 50 MeV helium ions. Our results also suggest that the considerable amount of DNA lesions induced by $$gamma$$-rays and 50 MeV helium ions are repaired by non-homologous end-joining (NHEJ) repair mechanism, but the majority of DNA lesions induced by 220 MeV carbon ions are not repaired by NHEJ.


Molecular analysis of carbon ion induced mutations in yeast ${it Saccharomyces cerevisiae}$ cells

清水 喜久雄*; 松尾 陽一郎*; 泉 佳伸*; 長谷 純宏; 野澤 樹; 坂本 綾子; 鳴海 一成

JAEA-Review 2010-065, JAEA Takasaki Annual Report 2009, P. 79, 2011/01

To elucidate the molecular mechanism of mutagenesis caused by ion beam irradiation in yeast, two mutant strains ${it ogg1}$ and ${it msh2}$ which are deficient in mismatch repair mechanisms were used to measure mutation spectra. Several hot spots were found in the ${it ogg1}$ mutant, while mutations in the ${it msh2}$ mutant were distributed evenly for base substitution except one hot spot at position 345. These results suggest that the incorporation of damaged nucleotides was not uniform in yeast cells.


Role of AtPol$$zeta$$, AtRev1 and AtPol$$eta$$ in UV light-induced mutagenesis in Arabidopsis$$^{1[W]}$$

中川 繭*; 高橋 真哉*; 田中 淳; 鳴海 一成; 坂本 綾子

Plant Physiology, 155(1), p.414 - 420, 2011/01

 被引用回数:14 パーセンタイル:47.4(Plant Sciences)

損傷乗り越え複製(TLS)は、特殊なDNAポリメラーゼによって損傷DNAをバイパスすることによって複製障害を回避する機構である。われわれは高等植物における損傷乗り越え複製機構の役割を調べるため、TLS型DNAポリメラーゼを欠失させたシロイヌナズナの変異株における紫外線誘発突然変異頻度を解析した。AtPol$$zeta$$及びAtRev1を欠失させた植物では突然変異頻度は低下したことから、これらのポリメラーゼは突然変異を起こしやすいことがわかった。これに対し、AtPol$$eta$$を欠失した植物では突然変異頻度が上昇したことから、AtPol$$eta$$は突然変異を起こしにくいことがわかった。AtPol$$zeta$$とAtRev1の2重変異ではAtRev1の単独変異とほぼ同等の変異頻度であったのに対し、AtPol$$zeta$$とAtPol$$eta$$の2重変異では、AtPol$$eta$$で見られた高い変異頻度がほぼ失われた。このことから、AtPol$$zeta$$ and AtRev1は同一の経路で働くのに対し、AtPol$$eta$$はこれらとは別の経路で働くことが示唆された。


Molecular analysis of carbon ion induced mutations in the yeast ${it ogg1}$ and ${it msh2}$ mutants

松尾 陽一郎*; 西嶋 茂宏*; 長谷 純宏; 野澤 樹; 坂本 綾子; 鳴海 一成; 清水 喜久雄*

JAEA-Review 2009-041, JAEA Takasaki Annual Report 2008, P. 75, 2009/12

本研究では、真核生物の一種である出芽酵母の野生株、塩基除去修復が不活性であるogg1株及びミスマッチ修復が不活性であるmsh2株を用いて、炭素イオンビーム照射で誘発される突然変異について、URA3遺伝子の突然変異を検出する5-FOAによる選択系で、変異スペクトルの解析を行った。その結果、野生株及びogg1株ともに塩基置換の頻度が高く、特にogg1株では変異のすべてが塩基置換であった。また、msh2株では、一塩基欠失が全体の突然変異の大部分を占め、その中でもGC to TAのトランスバージョン変異が多く誘発されることが確認された。これらの結果から、8-oxoGの生成がイオンビームに起因する突然変異をおもに誘導し、OGG1及びMSH2遺伝子が遺伝子の安定性に強く貢献していることが示唆された。


A UVB-hypersensitive mutant in ${it Arabidopsis thaliana}$ is defective in the DNA damage response

坂本 綾子; Lan, V. T. T.*; Puripunyavanich, V.*; 長谷 純宏; 横田 裕一郎; 鹿園 直哉; 中川 繭*; 鳴海 一成; 田中 淳

Plant Journal, 60(3), p.509 - 517, 2009/07

 被引用回数:19 パーセンタイル:54.1(Plant Sciences)

細胞分裂周期の進行は、内的又は外的なさまざまな要因によって常にストレスを受けており、それによってDNA複製の中断や染色体の分配異常などが引き起こされることが知られている。細胞周期チェックポイントとは、こうした異常事態の際に細胞周期の進行を停止し、適切な処理が行われるまで次のステップに移行しないようにするための機構である。われわれは、シロイヌナズナの紫外線感受性変異株のスクリーニングの過程で、${it suv2}$ (sensitive to UV 2)変異株を単離した。${it suv2}$変異株は、さまざまなDNA変異原やヒドロキシウレアに対して感受性を示し、その表現系は損傷チェックポイントにかかわるAtATRの欠損株に非常によく似ていた。さらに、SUV2蛋白質はGCN4型のコイルドコイルドメインを持つ蛋白質をコードしており、N末側にはPI3K様プロテインキナーゼのターゲット配列が2か所存在していた。そこで、SUV2蛋白質同士どうしの相互作用を酵母two-hybridの系で解析したところ、${it suv2}$は2量体を形成することが明らかになった。以上の結果から、われわれは${it suv2}$がATRの活性を制御するATRIPのシロイヌナズナにおけるホモログであると結論づけ、AtATRIPと命名した。


Functional analysis of the low-fidelity DNA polymerase AtREV1

高橋 真哉*; 中川 繭; 田中 淳; 鳴海 一成; 清水 喜久雄*; 坂本 綾子

JAEA-Review 2008-055, JAEA Takasaki Annual Report 2007, P. 58, 2008/11

植物の紫外線耐性機構の研究過程で、YファミリーDNAポリメラーゼをコードしている新規遺伝子${it AtREV1}$を同定した。YファミリーDNAポリメラーゼは複製忠実度が低く、さまざまなDNA損傷をバイパスすると考えられている。${it AtREV1}$遺伝子欠損植物は、紫外線やDNA架橋剤に感受性を示す。${it AtREV1}$遺伝子産物であるAtREV1タンパク質の生化学的な機能を解析するために、組換え大腸菌を用いてAtREV1タンパク質を発現・精製し、精製したタンパク質を用いてデオキシヌクレオチド転移活性を調べた。その結果、AtREV1タンパク質は複製忠実度が低く、鋳型の塩基にかかわらず、dCMPを好んで挿入することがわかった。また、APサイトの相補鎖には塩基を挿入できるが、紫外線損傷塩基の相補鎖には塩基を挿入できなかった。AtREV1タンパク質の低い複製忠実度が突然変異を引き起こすかどうかを調べるために、野生株と${it AtREV1}$遺伝子欠損株の突然変異頻度を測定した。その結果、AtREV1タンパク質が紫外線あるいは$$gamma$$線で生じるDNA損傷の誤複製を起こすことで、突然変異誘発を促進していることがわかった。

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