Issay Narumi

PprI : a general switch responsible for extreme radioresistance of Deinococcus radiodurans

Deinococcus radiodurans exhibits an extraordinary ability to withstand the lethal and mutagenic effects of DNA damaging agents, particularly, ionizing radiation. Available evidence indicates that efficient repair of DNA damage and protection of the chromosomal structure are mainly responsible for the radioresistance. Little is known about the biochemical basis for this phenomenon. We have identified a unique gene, pprI, as a general switch for downstream DNA repair and protection pathways, from a natural mutant, in which pprI is disrupted by a transposon. Complete functional disruption of the gene in wild-type leads to dramatic sensitivity to ionizing radiation. Radioresistance of the disruptant could be fully restored by complementation with pprI. In response to radiation stress, PprI can significantly and specifically induce the gene expression of recA and pprA and enhance the enzyme activities of catalases. These results strongly suggest that PprI plays a crucial role in regulating multiple DNA repair and protection pathways in response to radiation stress.

PprA: a novel protein from Deinococcus radiodurans that stimulates DNA ligation.

The extraordinary radiation resistance of Deinococcus radiodurans results from the efficient capacity of the bacterium to repair DNA double‐strand breaks. By analysing the DNA damage repair‐deficient mutant, KH311, a unique radiation‐inducible gene (designated pprA) responsible for loss of radiation resistance was identified. Investigations in vitro showed that the gene product of pprA (PprA) preferentially bound to double‐stranded DNA carrying strand breaks, inhibited Escherichia coli exonuclease III activity, and stimulated the DNA end‐joining reaction catalysed by ATP‐dependent and NAD‐dependent DNA ligases. These results suggest that D. radiodurans has a radiation‐induced non‐homologous end‐joining repair mechanism in which PprA plays a critical role.

Metabolic profiling and cytological analysis of proanthocyanidins in immature seeds of Arabidopsis thaliana flavonoid accumulation mutants

Arabidopsis TRANSPARENT TESTA19 (TT19) encodes a glutathione-S-transferase (GST)-like protein that is involved in the accumulation of proanthocyanidins (PAs) in the seed coat. PA accumulation sites in tt19 immature seeds were observed as small vacuolar-like structures, whereas those in tt12, a mutant of the tonoplast-bound transporter of PAs, and tt12 tt19 were observed at peripheral regions of small vacuoles. We found that tt19 immature seeds had small spherical structures showing unique thick morphology by differential interference contrast microscopy. The distribution pattern of the thick structures overlapped the location of PA accumulation sites, and the thick structures were outlined with GFP-TT12 proteins in tt19. PA analysis showed higher (eightfold) levels of solvent-insoluble PAs in tt19 immature seeds compared with the wild type. Metabolic profiling of the solvent-soluble fraction by LC-MS demonstrated that PA derivatives such as epicatechins and epicatechin oligomers, although highly accumulated in the wild type, were absent in tt19. We also revealed that tt12 specifically accumulated glycosylated epicatechins, the putative transport substrates for TT12. tt12 tt19 showed a similar metabolic profile to tt19. Given the cytosolic localization of functional GFP-TT19 proteins, our results suggest that TT19, which acts prior to TT12, functions in the cytosol to maintain the regular accumulation of PA precursors, such as epicatechin and glycosylated epicatechin, in the vacuole. The PA pathway in the Arabidopsis seed coat is discussed in relation to the subcellular localization of PA metabolites.

Unlocking radiation resistance mechanisms: still a long way to go

Abstract Recent transcriptome analysis revealed that Deinococcus radiodurans efficiently coordinate their recovery from ionizing radiation through a complex network of DNA repair and metabolic pathway switching. However, the additional discovery of numerous irradiation-response genes has provided new targets for the identification of genes primarily crucial to radiation resistance. Investigations based on electron microscopy suggest that the observed radiation resistance in D. radiodurans might be partly caused by the presence of an unusual ring-like conformation of nucleoids. Although such investigations provide useful insights into the mechanisms underlying radiation resistance, a more detailed empirical explanation of why D. radiodurans is so radiation resistant is still needed. Further research based on alternative genetic and biochemical approaches should help to gain a better understanding of the mechanisms involved in DNA repair.

Genetic Characterization of Mutants Resistant to the Antiauxin p-Chlorophenoxyisobutyric Acid Reveals That AAR3, a Gene Encoding a DCN1-Like Protein, Regulates Responses to the Synthetic Auxin 2,4-Dichlorophenoxyacetic Acid in Arabidopsis Roots

To isolate novel auxin-responsive mutants in Arabidopsis (Arabidopsis thaliana), we screened mutants for root growth resistance to a putative antiauxin, p-chlorophenoxyisobutyric acid (PCIB), which inhibits auxin action by interfering the upstream auxin-signaling events. Eleven PCIB-resistant mutants were obtained. Genetic mapping indicates that the mutations are located in at least five independent loci, including two known auxin-related loci, TRANSPORT INHIBITOR RESPONSE1 and Arabidopsis CULLIN1. antiauxin-resistant mutants (aars) aar3-1, aar4, and aar5 were also resistant to 2,4-dichlorophenoxyacetic acid as shown by a root growth assay. Positional cloning of aar3-1 revealed that the AAR3 gene encodes a protein with a domain of unknown function (DUF298), which has not previously been implicated in auxin signaling. The protein has a putative nuclear localization signal and shares homology with the DEFECTIVE IN CULLIN NEDDYLATION-1 protein through the DUF298 domain. The results also indicate that PCIB can facilitate the identification of factors involved in auxin or auxin-related signaling.

Posttranslational modification of the umuD-encoded subunit of Escherichia coli DNA polymerase V regulates its interactions with the β processivity clamp

The Escherichia coli umuDC (pol V) gene products participate in both a DNA damage checkpoint control and translesion DNA synthesis. Interactions of the two umuD gene products, the 139-aa UmuD and the 115-aa UmuD′ proteins, with components of the replicative DNA polymerase (pol III), are important for determining which biological role the umuDC gene products will play. Here we report our biochemical characterizations of the interactions of UmuD and UmuD′ with the pol III β processivity clamp. These analyses demonstrate that UmuD possesses a higher affinity for β than does UmuD′ because of the N-terminal arm of UmuD (residues 1–39), much of which is missing in UmuD′. Furthermore, we have identified specific amino acid residues of UmuD that crosslink to β with p-azidoiodoacetanilide, defining the domain of UmuD important for the interaction. We have recently proposed a model for the solution structure of UmuD2 in which the N-terminal arm of each protomer makes extensive contacts with the C-terminal globular domain of its intradimer partner, masking part of each surface. Taken together, our findings suggest that UmuD2 has a higher affinity for the β-clamp than does UmuD′2 because of the structures of its N-terminal arms. Viewed in this way, posttranslational modification of UmuD, which entails the removal of its N-terminal 24 residues to yield UmuD′, acts in part to attenuate the affinity of the umuD gene product for the β-clamp. Implications of these structure–function analyses for the checkpoint and translesion DNA synthesis functions of the umuDC gene products are discussed.

Limited concentration of RecA delays DNA double- strand break repair in Deinococcus radiodurans R1

To evaluate the importance of RecA in DNA double‐strand break (DSB) repair, we examined the effect of low and high RecA concentrations such as 2500 and 100 000 molecules per cell expressed from the inducible Pspac promoter in Deinococcus radiodurans in absence or in presence of IPTG respectively. We showed that at low concentration, RecA has a negligible effect on cell survival after γ‐irradiation when bacteria were immediately plated on TGY agar whereas it significantly decreased the survival to γ‐irradiation of ΔddrA cells while overexpression of RecA can partially compensate the loss of DdrA protein. In contrast, when cells expressing limited concentration of RecA were allowed to recover in TGY2X liquid medium, they showed a delay in mending DSB, failed to reinitiate DNA replication and were committed to die during incubation. A deletion of irrE resulted in sensitivity to γ‐irradiation and mitomycin C treatment. Interestingly, constitutive high expression of RecA compensates partially the ΔirrE sensitization to mitomycin C. The cells with low RecA content also failed to cleave LexA after DNA damage. However, neither a deletion of the lexA gene nor the expression of a non‐cleavable LexA(Ind–) mutant protein had an effect on survival or kinetics of DNA DSB repair compared with their lexA+ counterparts in recA+ as well as in bacteria expressing limiting concentration of RecA, suggesting an absence of relationship between the absence of LexA cleavage and the loss of viability or the delay in the kinetics of DSB repair. Thus, LexA protein seems to play no major role in the recovery processes after γ‐irradiation in D. radiodurans.

Characterization of Pathways Dependent on the uvsE, uvrA1, or uvrA2 Gene Product for UV Resistance in Deinococcus radiodurans

The genome of a radiation-resistant bacterium, Deinococcus radiodurans, contains one uvsE gene and two uvrA genes, uvrA1 and uvrA2. Using a series of mutants lacking these genes, we determined the biological significance of these components to UV resistance. The UV damage endonuclease (UvsE)-dependent excision repair (UVER) pathway and UvrA1-dependent pathway show some redundancy in their function to counteract the lethal effects of UV. Loss of these pathways does not cause increased sensitivity to UV mutagenesis, suggesting either that these pathways play no function in inducing mutations or that there are mechanisms to prevent mutation other than these excision repair pathways. UVER efficiently removes both cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) from genomic DNA. In contrast, the UvrA1 pathway does not significantly contribute to the repair of CPDs but eliminates 6-4PPs. Inactivation of uvrA2 does not result in a deleterious effect on survival, mutagenesis, or the repair kinetics of CPDs and 6-4PPs, indicating a minor role in resistance to UV. Loss of uvsE, uvrA1, and uvrA2 reduces but does not completely abolish the ability to eliminate CPDs and 6-4PPs from genomic DNA. The result indicates the existence of a system that removes UV damage yet to be identified.

Deinococcus aerius sp. nov., isolated from the high atmosphere

An orange-pigmented, non-motile, coccoid bacterial strain, designated TR0125T, was isolated from dust samples collected in the high atmosphere above Japan. Phylogenetic analysis based on 16S rRNA gene sequences showed that the strain was within the radiation of Deinococcus species. The major peptidoglycan amino acids were D-glutamic acid, glycine, D-alanine, L-alanine and ornithine. The predominant fatty acids were iso-C17:0, iso-C17:1omega9c and iso-C15:0. Strong resistance to desiccation, UV-C and gamma radiation and high DNA G+C content also supported the affiliation of strain TR0125T to the genus Deinococcus. Strain TR0125T showed the highest 16S rRNA gene sequence similarity value (95.7%) to the type strain of Deinococcus apachensis, and phylogenetic analysis showed that it was further separated from D. apachensis than from Deinococcus geothermalis, indicating that strain TR0125T was not a member of these two Deinococcus species. In addition, phenotypic differences were found between strain TR0125T and the type strains of these two Deinococcus species. Therefore, a novel species of the genus Deinococcus, Deinococcus aerius sp. nov. (type strain, TR0125T=JCM 11750T=DSM 21212T), is proposed to accommodate this isolate.

The Deinococcus radiodurans uvrA gene: identification of mutation sites in two mitomycin-sensitive strains and the first discovery of insertion sequence element from deinobacteria

Deinococcus radiodurans (Dr) possesses a prominent ability to repair the DNA injury induced by various DNA-damaging agents including mitomycin C (MC), ultraviolet light (UV) and ionizing radiation. DNA damage resistance was restored in MC sensitive (MC(S)) mutants 2621 and 3021 by transforming with DNAs of four cosmid clones derived from the gene library of strain KD8301, which showed wild type (wt) phenotype to DNA-damaging agents. Gene affected by mutation (mtcA or mtcB) in both mutants was cloned and its nucleotide (nt) sequence was determined. The deduced amino acid (aa) sequence of the gene product consists of 1016 aa and shares homology with many bacterial UvrA proteins. The mutation sites of both mutants were identified by analyzing the polymerase chain reaction (PCR) fragments derived from the genomic DNA of the mutants. A 144-base pair (bp) deletion including the start codon for the uvrA gene was observed in DNA of the mutant 3021, causing a defect in the gene. On the other hand, an insertion sequence (IS) element intervened in the uvrA gene of the mutant 2621, suggesting the insertional inactivation of the gene. The IS element comprises 1322-bp long, flanked by 19-bp inverted terminal repeats (ITR), and generated a 6-bp target duplication (TD). Two open reading frames (ORFs) were found in the IS element. The deduced aa sequences of large and small ORFs show homology to a putative transposase found in IS4 of Escherichia coli (Ec) and to a resolvase found in ISXc5 of Xanthomonas campestris (Xc), respectively. This is the first discovery of IS element in deinobacteria, and the IS element was designated IS2621.