DNA bipyrimidine photoproduct repair and transcriptional response of UV-C irradiated <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Vegetative wild-type and DNA repair-deficient (homologous recombination, recA and nucleotide excision repair, uvrB) Bacillus subtilis cells were exposed to UV-C radiation. Colony formation, DNA bipyrimidine photoproducts and gene expression were measured during cell recovery. Gene expression was measured after 60 min cell recovery where 50% (wild-type), 30% (recA) and 8% (uvrB), respectively, of the UV-C induced DNA photoproducts were repaired. We examined changes in the gene expression following UV exposure in wild-type and both repair-deficient strains. A set of known and unknown genes were found to be significantly up-regulated in wild-type B. subtilis cells, whereas no or lower gene induction was determined for both mutant strains. In addition, the possible roles of newly identified UV-responsive genes are discussed with respect to cellular recovery following exposure to UV irradiation.     

Roles of PprA,IrrE, and RecA in the resistance of <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis> to germicidal and environmentally relevant UV radiation

To study the role of different DNA repair genes in the resistance of Deinococcus radiodurans to mono- and polychromatic UV radiation, wild-type strain and knockout mutants in RecA, PprA, and IrrE of D. radiodurans were irradiated with UV-C (254 nm), UV-(A + B) (280–400 nm) and UV-A (315–400 nm) radiation, and survival was monitored. The strain deficient in recA was highly sensitive to UV-C radiation compared to the wild-type, but showed no loss of resistance against irradiation with UV-(A + B) and UV-A, while pprA and irrE-deficient strains exhibited elevated sensitivity to UV-A and UV-(A + B) radiation. These results suggest that the repair of DNA double-strand breaks is essential after treatment with highly energetic UV-C radiation, whereas protection from oxidative stress may play a greater role in resistance to environmentally relevant UV radiation.     

拟南芥中myo-肌醇-多磷酸合成与代谢及其信号

在酵母、真菌、动物和植物等真核生物中, 以myo-肌醇为基石通过不同位点的磷酸化形成各种myo-肌醇-多磷酸及其衍生物。过去10年的研究发现这些肌醇多磷酸参与了膜脂定向转运、蛋白结构稳定、离子通道调控、RNA转运以及DNA修复和染色质重塑等细胞生物学的基本进程。近些年在模式植物拟南芥(Arabidopsis thaliana)的研究中, 许多调控植物生长发育和环境胁迫应答的重要基因被发现, 并证实这些基因参与myo-肌醇-多磷酸的合成与代谢。该文概述了拟南芥中myo-肌醇-多磷酸合成与代谢的基因调控机理, 综述了不同肌醇多磷酸作为信号分子的功能, 提出肌醇多磷酸如同一类信息代码传递着植物细胞有序进程的基本指令。     

拟南芥组蛋白分子伴侣AtHIRA参与体细胞同源重组及盐胁迫响应

HIRA是组蛋白H3.3的特异分子伴侣, 在组蛋白H3.3掺入染色质的过程中发挥重要作用。研究表明, HIRA在哺乳动物胚胎发育和DNA损伤修复过程中不可或缺。而目前人们对于植物中HIRA同源基因功能的研究相对较少。该研究主要关注拟南芥(Arabidopsis thaliana) AtHIRA基因在植物体细胞同源重组以及减数分裂同源重组过程中的功能。将体细胞同源重组和减数分裂同源重组报告系统分别导入野生型和hira-1突变体后统计同源重组频率, 结果表明在正常生长条件下及在伯莱霉素(bleomycin)或UV-C处理条件下, hira-1突变体体细胞的分子内和分子间同源重组频率均低于野生型。而在正常生长条件下, 野生型与hira-1突变体花粉母细胞间的减数分裂同源重组频率没有明显差异, hira-1突变体的DNA损伤水平与野生型接近。qRT-PCR结果表明, DNA损伤修复相关基因RAD51和RAD54在hira-1突变体中的表达水平均高于野生型。此外, 盐胁迫处理实验表明, hira-1突变体对于高盐胁迫更加敏感。综上, AtHIRA在拟南芥体细胞同源重组及盐胁迫响应过程中发挥了一定作用。     

A comparison of two DNA base excision repair glycosylases from Arabidopsis thaliana

Plants contain the genes for both formamidopyrimidine-DNA glycosylase (FPG) and oxoguanine glycosylase (OGG). These enzymes play analogous roles in the base excision repair pathways of bacteria (FPG) and archaea, yeast, and mammals (OGG). Why have plants retained both genes? We tested one hypothesis by comparing the specificities of Arabidopsis FPG and OGG purified from Escherichia coli expression clones. Using depurinated DNA as substrate, the specific activity of Arabidopsis FPG was higher than that of Arabidopsis OGG. Using DNA oxidized by treatment with light in the presence of methylene blue, the specific activities of Arabidopsis FPG and OGG were equal. Using an oligonucleotide containing one oxoguanine (paired with C) and labeled with fluorescein, the specific activity of Arabidopsis OGG was greater than that of either FPG. The results support the hypothesis that genes for the two enzymes have been retained during evolution of plants for their specialized enzyme activities.     

Altered somatic mutation level and DNA repair gene expression in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> exposed to ultraviolet C,salt, and cadmium stresses

It is known that somatic mutations arising during animal growth and ageing contribute to the development of neurodegenerative and other animal diseases. For plants, several studies showed that small-scale somatic DNA mutations accumulated during Arabidopsis life cycle. However, there is a lack of data on the influence of environmental stresses on somatic DNA mutagenesis in plants. In this study, we analyzed the effects of ultraviolet C (UV-C) irradiation, high soil salinity, and cadmium (CdI₃) stresses on the level of small-scale somatic DNA mutations in Arabidopsis thaliana. The number of DNA mutations was examined in the Actin2 3′UTR (Actin-U1), ITS1-5.8rRNA-ITS2 (ITS), and ribulose-1,5-biphosphate carboxylase/oxygenase (rbcL) DNA regions. We found that somatic mutation levels considerably increased in CdI₃-treated Arabidopsis plants, while the mutation levels declined in the UV-C- and NaCl-treated A. thaliana. Cadmium is a mutagen that is known to inhibit DNA repair processes. The detected stress-induced alterations in somatic DNA mutation levels were accompanied by markedly increased expression of base excision repair genes (AtARP, AtDME, AtDML2, AtDML3, AtMBD4, AtROS, AtUNG, and AtZDP), nucleotide excision repair genes (AtDDB1a, AtRad4, and AtRad23a), mismatch repair genes (AtMSH2, AtMSH3, and AtMSH7), and photoreactivation genes (AtUVR2, AtUVR3). Thus, the results demonstrated that UV-C, high soil salinity, and cadmium stresses influence both the level of DNA mutations and expression of DNA repair genes. Salt- and UV-induced activation of DNA repair genes could contribute to the stress-induced decrease in somatic mutation level.     

CENTRIN2 Interacts with the Arabidopsis Homolog of the Human XPC Protein (AtRAD4) and Contributes to Efficient Synthesis-dependent Repair of Bulky DNA Lesions

Arabidopsis thaliana CENTRIN2 (AtCEN2) has been shown to modulate Nucleotide Excision Repair (NER) and Homologous Recombination (HR). The present study provides evidence that AtCEN2 interacts with the Arabidopsis homolog of human XPC, AtRAD4 and that the distal EF-hand Ca²⁺ binding domain is essential for this interaction. In addition, the synthesis-dependent repair efficiency of bulky DNA lesions was enhanced in cell extracts prepared from Arabidopsis plants overexpressing the full length AtCEN2 but not in those overexpressing a truncated AtCEN2 form, suggesting a role for the distal EF-hand Ca²⁺ binding domain in the early step of the NER process. Upon UV-C treatment the AtCEN2 protein was shown to be increased in concentration and to be localised in the nucleus rapidly. Taken together these data suggest that AtCEN2 is a part of the AtRAD4 recognition complex and that this interaction is required for efficient NER. In addition, NER and HR appear to be differentially modulated upon exposure of plants to DNA damaging agents. This suggests in plants, that processing of bulky DNA lesions highly depends on the excision repair efficiency, especially the recognition step, thus influencing the recombinational repair pathway.     

Comparative DNA Damage and Repair in Echinoderm Coelomocytes Exposed to Genotoxicants

The capacity to withstand and repair DNA damage differs among species and plays a role in determining an organism''s resistance to genotoxicity, life history, and susceptibility to disease. Environmental stressors that affect organisms at the genetic level are of particular concern in ecotoxicology due to the potential for chronic effects and trans-generational impacts on populations. Echinoderms are valuable organisms to study the relationship between DNA repair and resistance to genotoxic stress due to their history and use as ecotoxicological models, little evidence of senescence, and few reported cases of neoplasia. Coelomocytes (immune cells) have been proposed to serve as sensitive bioindicators of environmental stress and are often used to assess genotoxicity; however, little is known about how coelomocytes from different echinoderm species respond to genotoxic stress. In this study, DNA damage was assessed (by Fast Micromethod) in coelomocytes of four echinoderm species (sea urchins Lytechinus variegatus, Echinometra lucunter lucunter, and Tripneustes ventricosus, and a sea cucumber Isostichopus badionotus) after acute exposure to H₂O₂ (0–100 mM) and UV-C (0–9999 J/m²), and DNA repair was analyzed over a 24-hour period of recovery. Results show that coelomocytes from all four echinoderm species have the capacity to repair both UV-C and H₂O₂-induced DNA damage; however, there were differences in repair capacity between species. At 24 hours following exposure to the highest concentration of H₂O₂ (100 mM) and highest dose of UV-C (9999 J/m²) cell viability remained high (>94.6±1.2%) but DNA repair ranged from 18.2±9.2% to 70.8±16.0% for H₂O₂ and 8.4±3.2% to 79.8±9.0% for UV-C exposure. Species-specific differences in genotoxic susceptibility and capacity for DNA repair are important to consider when evaluating ecogenotoxicological model organisms and assessing overall impacts of genotoxicants in the environment.     

Evidence for a Role of Arabidopsis CDT1 Proteins in Gametophyte Development and Maintenance of Genome Integrity

Meristems retain the ability to divide throughout the life cycle of plants, which can last for over 1000 years in some species. Furthermore, the germline is not laid down early during embryogenesis but originates from the meristematic cells relatively late during development. Thus, accurate cell cycle regulation is of utmost importance to avoid the accumulation of mutations during vegetative growth and reproduction. The Arabidopsis thaliana genome encodes two homologs of the replication licensing factor CDC10 Target1 (CDT1), and overexpression of CDT1a stimulates DNA replication. Here, we have investigated the respective functions of Arabidopsis CDT1a and CDT1b. We show that CDT1 proteins have partially redundant functions during gametophyte development and are required for the maintenance of genome integrity. Furthermore, CDT1-RNAi plants show endogenous DNA stress, are more tolerant than the wild type to DNA-damaging agents, and show constitutive induction of genes involved in DNA repair. This DNA stress response may be a direct consequence of reduced CDT1 accumulation on DNA repair or may relate to the ability of CDT1 proteins to form complexes with DNA polymerase ε, which functions in DNA replication and in DNA stress checkpoint activation. Taken together, our results provide evidence for a crucial role of Arabidopsis CDT1 proteins in genome stability.     

Identification of promoters bound by c-Jun/ATF2 during rapid large-scale gene activation following genotoxic stress

The NH2-terminal Jun kinases (JNKs) function in diverse roles through phosphorylation and activation of AP-1 components including ATF2 and c-Jun. However, the genes that mediate these processes are poorly understood. A model phenotype characterized by rapid activation of Jun kinase and enhanced DNA repair following cisplatin treatment was examined using chromatin immunoprecipitation with antibodies against ATF2 and c-Jun or their phosphorylated forms and hybridization to promoter arrays. Following genotoxic stress, we identified 269 genes whose promoters are bound upon phosphorylation of ATF2 and c-Jun. Binding did not occur following treatment with transplatin or the JNK inhibitor SP600125 or JNK-specific siRNA. Of 89 known DNA repair genes represented on the array, 23 are specifically activated by cisplatin treatment within 3-6 hr. Thus, the genotoxic stress response occurs at least partly via activation of ATF2 and c-Jun, leading to large-scale coordinate gene expression dominated by genes of DNA repair.     

The RecQ gene family in plants

RecQ helicases are conserved throughout all kingdoms of life regarding their overall structure and function. They are 3'-5' DNA helicases resolving different recombinogenic DNA structures. The RecQ helicases are key factors in a number of DNA repair and recombination pathways involved in the maintenance of genome integrity. In eukaryotes the number of RecQ genes and the structure of RecQ proteins vary strongly between organisms. Therefore, they have been named RecQ-like genes. Knockouts of several RecQ-like genes cause severe diseases in animals or harmful cellular phenotypes in yeast. Until now the largest number of RecQ-like genes per organism has been found in plants. Arabidopsis and rice possess seven different RecQ-like genes each. In the almost completely sequenced genome of the moss Physcomitrella patens at least five RecQ-like genes are present. One of the major present and future research aims is to define putative plant-specific functions and to assign their roles in DNA repair and recombination pathways in relation to RecQ genes from other eukaryotes. Regarding their intron positions, the structures of six RecQ-like genes of dicots and monocots are virtually identical indicating a conservation over a time scale of 150 million years. In contrast to other eukaryotes one gene (RecQsim) exists exclusively in plants. It possesses an interrupted helicase domain but nevertheless seems to have maintained the RecQ function. Owing to a recent gene duplication besides the AtRecQl4A gene an additional RecQ-like gene (AtRecQl4B) exists in the Brassicaceae only. Genetic studies indicate that a AtRecQl4A knockout results in sensitivity to mutagens as well as an hyper-recombination phenotype. Since AtRecQl4B was still present, both genes must have non-redundant roles. Analysis of plant RecQ-like genes will not only increase the knowledge on DNA repair and recombination, but also on the evolution and radiation of protein families.     

An SMC-like protein is required for efficient homologous recombination in Arabidopsis.

In plants, the observed low frequency of gene targeting and intrachromosomal recombination contrasts markedly with the efficient extrachromosomal recombination of DNA. Thus, chromatin accessibility can have a major influence on the recombination frequency of chromosomal DNA in vivo. An Arabidopsis mutant hypersensitive to a range of DNA-damaging treatments (UV-C, X-rays, methyl methanesulfonate and mitomycin C) is also defective in somatic intrachromosomal homologous recombination. The wild-type gene encodes a protein closely related to the structural maintenance of chromosomes (SMC) family involved in structural changes in chromosomes. Although loss of SMC function is lethal in other eukaryotes, growth of the Arabidopsis mutant is normal in the absence of genotoxic treatments. This suggests a surprisingly specialized function for this protein in plants, and provides the first in vivo evidence for the involvement of an SMC protein in recombinational DNA repair. It is possible that SMC-like proteins in plants alleviate suppressive chromatin structure limiting homologous recombination in somatic cells.