Ayako N. Sakamoto

An Ultraviolet-B-Resistant Mutant with Enhanced DNA Repair in Arabidopsis

An ultraviolet-B (UV-B)-resistant mutant,uvi1 (UV-B insensitive 1), of Arabidopsis was isolated from 1,280 M1 seeds that had been exposed to ion beam irradiation. The fresh weight ofuvi1 under high-UV-B exposure was more than twice that of the wild type. A root-bending assay indicated that root growth was less inhibited by UV-B exposure in uvi1 than in the wild type. When the seedlings were grown under white light, the UV-B dose required for 50% inhibition was about 6 kJ m−2for the wild type and 9 kJ m−2 for uvi1. When the seedlings were irradiated with UV-B in darkness, the dose required for 50% inhibition was about 1.5 kJ m−2 for the wild type and 4 kJ m−2 for uvi1. An enzyme-linked immunosorbent assay showed that the reduction in levels of cyclobutane pyrimidine dimers (CPDs) under white light and of (6-4) photoproducts in darkness occurred faster in uvi1 than in the wild type. These results indicate that uvi1 had increased photoreactivation of CPDs and dark repair of (6-4) photoproducts, leading to strong UV-B resistance. Furthermore, the transcript levels of PHR1 (CPD photolyase gene) were much higher inuvi1 than in the wild type both under white light and after UV-B exposure. Placing the plants in the dark before UV-B exposure decreases the early reduction of CPDs in the wild type but not inuvi1. Our results suggest that UVI1 is a negative regulator of two independent DNA repair systems.

Roles of Arabidopsis AtREV1 and AtREV7 in Translesion Synthesis

Plants have mechanisms for repairing and tolerating detrimental effects by various DNA damaging agents. A tolerance pathway that has been predicted to be present in higher plants is translesion synthesis (TLS), which is catalyzed by polymerases. In Arabidopsis (Arabidopsis thaliana), however, the only gene known to be involved in TLS is the Arabidopsis homolog of REV3, AtREV3, which is a putative catalytic subunit of Arabidopsis DNA polymerase ζ. A disrupted mutant of AtREV3, rev3, was previously found to be highly sensitive to ultraviolet-B (UV-B) and various DNA damaging agents. REV1 and REV7 are thought to be components of translesion synthesis in plants. In this study, we identified the Arabidopsis homologs of REV1 and REV7 (AtREV1 and AtREV7). Several mutants carrying disrupted AtREV1 and AtREV7 genes were isolated from Arabidopsis T-DNA-inserted lines. An AtREV1-disrupted mutant, rev1, was found to be moderately sensitive to UV-B and DNA cross-linkers. A rev1rev3 double mutant, like rev3, showed high sensitivity to UV-B, γ-rays, and DNA cross-linkers. An AtREV7-disrupted mutant, rev7, was possibly sensitive to cis-diamminedichloroplatinum(II), a kind of DNA cross-linker, but it was not sensitive to acute UV-B and γ-ray irradiation. On the other hand, the aerial growth of rev7, like the aerial growth of rev1 and rev3, was inhibited by long-term UV-B. These results suggest that a TLS mechanism exists in a higher plant and show that AtREV1 and AtREV7 have important roles in tolerating exposure to DNA-damaging agents.

Arabidopsis thaliana Y-family DNA polymerase η 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.

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.

AtREV1, a Y-Family DNA Polymerase in Arabidopsis, Has Deoxynucleotidyl Transferase Activity in Vitro

To clarify the functions of the Arabidopsis thaliana REV1 (AtREV1) protein, we expressed it in Escherichia coli and purified it to near homogeneity. The deoxynucleotidyl transferase activity of the recombinant AtREV1 was examined in vitro using a primer extension assay. The recombinant AtREV1 transferred one or two nucleotides to the primer end. It efficiently inserted dCMP regardless of the opposite base. AtREV1 also inserted a dCMP opposite an apurinic/apyrimidinic site, which is physiologically generated or induced by various DNA-damaging agents. In contrast, AtREV1 had no insertion activities against UV-inducible DNA lesions as reported in yeast or mammalian system. Although the substrate specificity of AtREV1 was rather narrow in the presence of magnesium ion, it widened in the presence of manganese ion. These results suggest that AtREV1 serves as a deoxycytidyl transferase in plant cells.

Mutational effects of γ-rays and carbon ion beams on Arabidopsis seedlings

To assess the mutational effects of radiation on vigorously proliferating plant tissue, the mutation spectrum was analyzed with Arabidopsis seedlings using the plasmid-rescue method. Transgenic plants containing the Escherichia coli rpsL gene were irradiated with γ-rays and carbon ion beams (320-MeV 12C6+), and mutations in the rpsL gene were analyzed. Mutant frequency increased significantly following irradiation by γ-rays, but not by 320-MeV 12C6+. Mutation spectra showed that both radiations increased the frequency of frameshifts and other mutations, including deletions and insertions, but only γ-rays increased the frequency of total base substitutions. These results suggest that the type of DNA lesions which cause base substitutions were less often induced by 320-MeV 12C6+ than by γ-rays in Arabidopsis seedlings. Furthermore, γ-rays never increased the frequencies of G:C to T:A or A:T to C:G transversions, which are caused by oxidized guanine; 320-MeV 12C6+, however, produced a slight increase in both transversions. Instead, γ-rays produced a significant increase in the frequency of G:C to A:T transitions. These results suggest that 8-oxoguanine has little effect on mutagenesis in Arabidopsis cells.

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

UVB radiation suppresses photosynthesis and protein biosynthesis in plants, which in turn decreases growth and productivity. Here, an ultraviolet-B (UVB)-tolerant rice mutant, utr319 (UV Tolerant Rice 319), was isolated from a mutagenized population derived from 2500 M1 seeds (of the UVB-resistant cultivar ‘Sasanishiki’) that were exposed to carbon ions. The utr319 mutant was more tolerant to UVB than the wild type. Neither the levels of UVB-induced cyclobutane pyrimidine dimers (CPDs) or (6-4) pyrimidine-pyrimidone photodimers [(6-4) photoproducts], nor the repair of CPDs or (6-4) photoproducts, was altered in the utr319 mutant. Thus, the utr319 mutant may be impaired in the production of a previously unidentified factor that confers UVB tolerance. To identify the mutated region in the utr319 mutant, microarray-based comparative genomic hybridization analysis was performed. Two adjacent genes on chromosome 7, Os07g0264900 and Os07g0265100, were predicted to represent the mutant allele. Sequence analysis of the chromosome region in utr319 revealed a deletion of 45 419 bp. RNAi analysis indicated that Os07g0265100 is most likely the mutated gene. Database analysis indicated that the Os07g0265100 gene, UTR319, encodes a putative protein with unknown characteristics or function. In addition, the homologs of UTR319 are conserved only among land plants. Therefore, 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.

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.

An ion beam–induced Arabidopsis mutant with marked chromosomal rearrangement

Abstract Ion beams have been used as an effective tool in mutation breeding for the creation of crops with novel characteristics. Recent analyses have revealed that ion beams induce large chromosomal alterations, in addition to small mutations comprising base changes or frameshifts. In an effort to understand the potential capability of ion beams, we analyzed an Arabidopsis mutant possessing an abnormal genetic trait. The Arabidopsis mutant uvh3-2 is hypersensitive to UVB radiation when photoreactivation is unavailable. uvh3-2 plants grow normally and produce seeds by self-pollination. SSLP and CAPS analyses of F2 plants showed abnormal recombination frequency on chromosomes 2 and 3. PCR-based analysis and sequencing revealed that one-third of chromosome 3 was translocated to chromosome 2 in uvh3-2. FISH analysis using a 180 bp centromeric repeat and 45S ribosomal DNA (rDNA) as probes showed that the 45S rDNA signal was positioned away from that of the 180 bp centromeric repeat in uvh3-2, suggesting the insertion of a large chromosome fragment into the chromosome with 45S rDNA clusters. F1 plants derived from a cross between uvh3-2 and wild-type showed reduced fertility. PCR-based analysis of F2 plants suggested that reproductive cells carrying normal chromosome 2 and uvh3-2–derived chromosome 3 are unable to survive and therefore produce zygote. These results showed that ion beams could induce marked genomic alterations, and could possibly lead to the generation of novel plant species and crop strains.

Editorial: Maintenance of Genome Integrity: DNA Damage Sensing, Signaling, Repair, and Replication in Plants

Because of their sessile lifestyles, plants are continuously exposed to DNA-damaging agents present in the environment. Although the basic mechanisms of genome maintenance are conserved between animal and plant kingdom, plants also have evolved specific mechanisms to cope with DNA damage. Indeed, studies in past decades have demonstrated the presence of elastic mechanisms in plants. For example, when exposed to DNA damaging agents, plants respond immediately to start repairing the damage, regulating cell proliferation, changing metabolic pathways. Here we are proud to have twelve outstanding articles focus on the maintenance of genome integrity: DNA damage sensing, signaling, repair and replication in plants. The present e-book is opened by several comprehensive reviews dealing with genomic and extra-genomic DNA maintenance, as well as the role of double strand break (DSB) signaling in plants. In his minireview, Roy provides an upgraded view on the link connecting chromatin structure stability and DNA damage response at the genetic and epigenetic levels, while Amiard et al. present an overview on DSB repair pathways in Arabidopsis thaliana, with focus on the signaling of DNA breaks and deprotected telomeres. Stability of genomic DNA, not only in nuclei, but also in organelles is crucial for plant development. In contrast to nuclear genomes, the amount and structural integrity of organellar genomes changes during plant development. It is very interesting to consider why organellar genomes are less stable as the replication and repair machineries are encoded by the nuclear genome, yet the cause of the instability is poorly understood. Oldenburg and Bendich first explain the history of the studies into the size and structure of organellar DNA (orgDNA). They then address the copy number and integrity of orgDNA during plant development. The review continues with an overview of the proteins which are involved in the processes of orgDNA replication, repair, and recombination, and changes in the amount of these proteins during leaf development. It has been observed that plastid DNA (ptDNA) maintenance in grasses differs from that in dicots as it rapidly declines upon light exposure. From these observations, the authors propose the idea that instead of repairing damaged DNA, grasses use a cost-saving involving a loss of ptDNA. DNA polymerases are crucial for maintenance of genome integrity in organisms. The family X DNA polymerases work in DNA repairs such as base excision repair (BER) and/or DSB repair. Plants are unique in having only one member of this family, polymerase lambda (Polλ). Furukawa et al. showed that the Polλ knockout Arabidopsis (atpolλ-1) is only mildly sensitive to DSB-inducing treatments, whereas the double-knockouts of AtPolλ and AtLig4 made the plants hypersensitive to DSB compared to each single knockout. These results suggest that the AtPolλ has a role in DSB repair, probably in an AtLig4-independent non-homologous end-joining (NHEJ) pathway. Proliferating cell nuclear antigen (PCNA) is a key component of eukaryotic DNA replication machinery. PCNA usually accompanies the DNA polymerase to gather a specific set of proteins onto the replication fork when replication is disturbed. Cyclin Ds are expressed in G2 and degraded during G2/M transition. When the checkpoint is activated, the degradation of cyclin D is inhibited to arrest cells at G2. Strzalka et al. demonstrated that Arabidopsis PCNAs directly interact with some members of the cyclin Ds in yeast and plant cells, suggesting that PCNAs link the signal of disturbed replication with cell cycle control. Ultraviolet (UV) light has been used to analyze cellular DNA-damage responses. UV induces cyclobutanate pyrimidine dimmers (CPDs) and other damage to DNA, which triggers various cellular responses: DNA repair, cell cycle delay or arrest, and cell death. Thus, UVB in sunlight can confer severe stress to plants, but plants have photorepair enzymes to correct the damage. Takahashi et al. investigated the responses of plant cells irradiated with low or high dose of UVB. UVB irradiated cells showed different reactions, depending on the dose, suggesting that accumulation of CPDs caused by high dose UVB induces formation of single or DSBs, which leads to cell death. In their research article, Questa et al. discuss on the roles of the DDM1 and ROS1 genes in UVB-induced DNA repair by using Arabidopsis mutants and a set of analytical measurements. Disruption of these genes had an opposite impact of these two genes, with the ddm1 mutants accumulating more DNA damage, while the ros1 mutants showed less amount of DNA damage. Based on experimental work, they hypothesize that the DNA demethylation in the ddm1 mutant can affect the accessibility of DNA repair systems in this region, while the better performance of ros1 mutants can arise as a result of increased levels of photolyases. In their research on the MAPK signal transduction in response to aluminum (Al) treatments, Panda and Achary underline the biphasic mode of action of Al-induced DNA damage in Allium cepa. They observed that at high concentrations Al induces DNA damage, while at low concentration an adaptive response is present, and hypothesize that the MAPK-DNA repair network is responsible for both actions. The role played by DNA/RNA helicases against the genotoxic effects of abiotic stress is documented. XPB (xeroderma pigmentosum type B) helicases promote nucleotide excision repair (NER) by unwinding double strand DNA at the damaged sites. In the attempt to assess the potential of plant XPB helicases as tools in counteracting adverse environmental conditions, Raikwar et al. carried out an in silico and functional characterization of the rice OsXPB2 gene promoter in response to abiotic stress and hormone-based treatments. Based on its multi-stress responsiveness, the OsXPB2 promoter represents a promising tool for improving the response of crops to genotoxic stress. Huefner et al. investigated the short- and long-term impact of high-LET (Linear Energy Transfer) HZE (high atomic weight, high energy) particles vs. low-LET gamma rays on genome stability, using Arabidopsis mutants defective in DNA repair and cell-cycle checkpoint. This study highlights the increased sensitivity of Arabidopsis plants to HZE radiation, revealing the predominant role played by ATR (ATM and Rad3-related) protein kinase compared to ATM (ataxia-telangiectasia mutated) in the response to high-LET radiation. In the accompanying article, Missirian et al. compared the transcriptional response in Arabidopsis seedlings exposed to HZE radiation vs. DSB-inducing agents as gamma rays, bleomycin and mitomycin C. These treatments triggered an intense, short-term DSB-specific repair response which was not detected in plants challenged with conventional stresses. A distinctive feature of the HZE transcriptional response was the early activation of key genes involved in the catabolism of cellular components. With genome editing being the cutting-edge topic of present days, the article by Cantos et al. deals with the implementation of such tool (zinc finger nucleases, ZFNs) for the identification of appropriate regions for safe gene insertion. By harnessing their ability to induce DSBs at the cutting site, ZFNs trigger the NHEJ or homologous recombination (HR) DNA repair pathways at the targeted site. This study used ZFNs with short DNA recognition domains, able to target multiple sites within the rice genome, and subsequently study the integration patterns of the GUS marker gene, allowing the identification of “safe harbors,” intergenic regions with potential high expression. The present e-book provides an up-date overview of the ongoing research dealing with different aspects of the DNA damage response in plants, highlighting the complexity of molecular networks involved in genome maintenance. A better understanding of DNA damage accumulation/perception/signaling/repair mechanisms in planta is expected to speed up crop improvement through conventional breeding and gene-transfer based techniques.