Regulation of UVR8 photoreceptor dimer/monomer photo-equilibrium in Arabidopsis plants grown under photoperiodic conditions.

The UV RESISTANCE LOCUS 8 (UVR8) photoreceptor specifically mediates photomorphogenic responses to UV-B. Photoreception induces dissociation of dimeric UVR8 into monomers to initiate responses. However, the regulation of dimer/monomer status in plants growing under photoperiodic conditions has not been examined. Here we show that UVR8 establishes a dimer/monomer photo-equilibrium in plants growing in diurnal photoperiods in both controlled environments and natural daylight. The photo-equilibrium is determined by the relative rates of photoreception and dark-reversion to the dimer. Experiments with mutants in REPRESSOR OF UV-B PHOTOMORPHOGENESIS 1 (RUP1) and RUP2 show that these proteins are crucial in regulating the photo-equilibrium because they promote reversion to the dimer. In plants growing in daylight, the UVR8 photo-equilibrium is most strongly correlated with low ambient fluence rates of UV-B (up to 1.5 µmol m(-2) s(-1) ), rather than higher fluence rates or the amount of photosynthetically active radiation. In addition, the rate of reversion of monomer to dimer is reduced at lower temperatures, promoting an increase in the relative level of monomer at approximately 8-10 °C. Thus, UVR8 does not behave like a simple UV-B switch under photoperiodic growth conditions but establishes a dimer/monomer photo-equilibrium that is regulated by UV-B and also influenced by temperature.

Reaction dynamics of the UV-B photosensor UVR8.

UVR8 is a recently discovered ultraviolet-B (UV-B) photoreceptor protein identified in plants and algae. In the dark state, UVR8 exists as a homodimer, whereas UV-B irradiation induces UVR8 monomerization and initiation of signaling. Although the biological functions of UVR8 have been studied, the fundamental reaction mechanism and associated kinetics have not yet been fully elucidated. Here, we used the transient grating method to determine the reaction dynamics of UVR8 monomerization based on its diffusion coefficient. We found that the UVR8 photodissociation reaction proceeds in three stages: (i) photoexcitation of cross-dimer tryptophan (Trp) pyramids; (ii) an initial conformational change with a time constant of 50 ms; and (iii) dimer dissociation with a time constant of 200 ms. We identified W285 as the key Trp residue responsible for initiating this photoreaction. Although the C-terminus of UVR8 is essential for biological interactions and signaling via downstream components such as COP1, no obvious differences were detected between the photoreactions of wild-type UVR8 (amino acids 1-440) and a mutant lacking the C-terminus (amino acids 1-383). This similarity indicates that the conformational change associated with stage ii cannot primarily be attributed to this region. A UV-B-driven conformational change with a time constant of 50 ms was also detected in the monomeric mutants of UVR8. Dimer recovery following monomerization, as measured by circular dichroism spectroscopy, was decreased under oxygen-purged conditions, suggesting that redox reactivity is a key factor contributing to the UVR8 oligomeric state.

UV-B detected by the UVR8 photoreceptor antagonizes auxin signaling and plant shade avoidance.

Plants detect different facets of their radiation environment via specific photoreceptors to modulate growth and development. UV-B is perceived by the photoreceptor UV RESISTANCE LOCUS 8 (UVR8). The molecular mechanisms linking UVR8 activation to plant growth are not fully understood, however. When grown in close proximity to neighboring vegetation, shade-intolerant plants initiate dramatic stem elongation to overtop competitors. Here we show that UV-B, detected by UVR8, provides an unambiguous sunlight signal that inhibits shade avoidance responses in Arabidopsis thaliana by antagonizing the phytohormones auxin and gibberellin. UV-B triggers degradation of the transcription factors PHYTOCHROME INTERACTING FACTOR 4 and PHYTOCHROME INTERACTING FACTOR 5 and stabilizes growth-repressing DELLA proteins, inhibiting auxin biosynthesis via a dual mechanism. Our findings show that UVR8 signaling is closely integrated with other photoreceptor pathways to regulate auxin signaling and plant growth in sunlight.

Photochemical reaction mechanism of UV-B-induced monomerization of UVR8 dimers as the first signaling event in UV-B-regulated gene expression in plants.

The Arabidopsis thaliana UV RESISTANCE LOCUS8 (UVR8) protein has been identified to specifically mediate photomorphogenic UV-B responses by acting as a UV-B photoreceptor. The dimeric structure of the UVR8 protein dissociates into signaling-active monomers upon UV-B exposure, and the monomers rapidly interact with downstream signaling components to regulate gene expression. UVR8 monomers revert to dimers in the absence of UV-B radiation, thereby reversing transcription activation. UVR8 amino acid residues W233 and W285 have been identified to play critical roles in the UVR8 dimer for the response to UV-B irradiation. In the present work, the photoreaction mechanism for UVR8 monomerization is explored with quantum chemical cluster calculations and evaluated by molecular dynamics simulations using the wild-type UVR8 dimer and novel force field parameters developed for intermediate radicals formed in the photochemical process. Three different models are investigated, which show that the preferred mechanism for UVR8 monomerization involves electron transfer from residue W233 to W285 and onward to R338 initiated by UV-B irradiation, coupled to simultaneous proton transfer from W233 to D129 leading to the formation of protonated D129, a deprotonated W233 radical, and a neutral R338 radical. Due to the formation of the neutral R338 radical, salt bridges involving this residue are disrupted together with the concomitant interruption of several other key salt bridges R286-D96, R286-D107, R338-D44, R354-E43, and R354-E53. The resulting large decrease in protein-protein interaction energy arising from this sequence of events leads to the monomerization of the UVR8 dimer. The mechanism presented is in accord with all experimental data available to date.

DNA-damage response in chromatin of ribosomal genes and the surrounding genome.

DNA repair events have functional significance especially for genome stability. Although the DNA damage response within the whole genome has been extensively studied, the region-specific characteristics of nuclear sub-compartments such as the nucleolus or fragile sites have not been fully elucidated. Here, we show that the heterochromatin protein HP1 and PML protein recognize spontaneously occurring 53BP1- or γ-H2AX-positive DNA lesions throughout the genome. Moreover, 53BP1 nuclear bodies, which co-localize with PML bodies, also occur within the nucleoli compartments. Irradiation of the human osteosarcoma cell line U2OS with γ-rays increases the degree of co-localization between 53BP1 and PML bodies throughout the genome; however, the 53BP1 protein is less abundant in chromatin of ribosomal genes and fragile sites (FRA3B and FRA16D) in γ-irradiated cells. Most epigenomic marks on ribosomal genes and fragile sites are relatively stable in both non-irradiated and γ-irradiated cells. However, H3K4me2, H3K9me3, H3K27me3 and H3K79me1 were significantly changed in promoter and coding regions of ribosomal genes after exposure of cells to γ-rays. In fragile sites, γ-irradiation induces a decrease in H3K4me3, changes the levels of HP1ß, and modifies the levels of H3K9 acetylation, while the level of H3K9me3 was relatively stable. In these studies, we confirm a specific DNA-damage response that differs between the ribosomal genes and fragile sites, which indicates the region-specificity of DNA repair.

In vivo function of tryptophans in the Arabidopsis UV-B photoreceptor UVR8.

Arabidopsis thaliana UV RESISTANCE LOCUS8 (UVR8) is a photoreceptor specifically for UV-B light that initiates photomorphogenic responses in plants. UV-B exposure causes rapid conversion of UVR8 from dimer to monomer, accumulation in the nucleus, and interaction with CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1), which functions with UVR8 in UV-B responses. Studies in yeast and with purified UVR8 implicate several tryptophan amino acids in UV-B photoreception. However, their roles in UV-B responses in plants, and the functional significance of all 14 UVR8 tryptophans, are not known. Here we report the functions of the UVR8 tryptophans in vivo. Three tryptophans in the ß-propeller core are important in maintaining structural stability and function of UVR8. However, mutation of three other core tryptophans and four at the dimeric interface has no apparent effect on function in vivo. Mutation of three tryptophans implicated in UV-B photoreception, W233, W285, and W337, impairs photomorphogenic responses to different extents. W285 is essential for UVR8 function in plants, whereas W233 is important but not essential for function, and W337 has a lesser role. Ala mutants of these tryptophans appear monomeric and constitutively bind COP1 in plants, but their responses indicate that monomer formation and COP1 binding are not sufficient for UVR8 function.

Structural basis of ultraviolet-B perception by UVR8.

The Arabidopsis thaliana protein UVR8 is a photoreceptor for ultraviolet-B. Upon ultraviolet-B irradiation, UVR8 undergoes an immediate switch from homodimer to monomer, which triggers a signalling pathway for ultraviolet protection. The mechanism by which UVR8 senses ultraviolet-B remains largely unknown. Here we report the crystal structure of UVR8 at 1.8 Å resolution, revealing a symmetric homodimer of seven-bladed ß-propeller that is devoid of any external cofactor as the chromophore. Arginine residues that stabilize the homodimeric interface, principally Arg 286 and Arg 338, make elaborate intramolecular cation-π interactions with surrounding tryptophan amino acids. Two of these tryptophans, Trp 285 and Trp 233, collectively serve as the ultraviolet-B chromophore. Our structural and biochemical analyses identify the molecular mechanism for UVR8-mediated ultraviolet-B perception, in which ultraviolet-B radiation results in destabilization of the intramolecular cation-π interactions, causing disruption of the critical intermolecular hydrogen bonds mediated by Arg 286 and Arg 338 and subsequent dissociation of the UVR8 homodimer.

Perception of UV-B by the Arabidopsis UVR8 protein.

To optimize their growth and survival, plants perceive and respond to ultraviolet-B (UV-B) radiation. However, neither the molecular identity of the UV-B photoreceptor nor the photoperception mechanism is known. Here we show that dimers of the UVR8 protein perceive UV-B, probably by a tryptophan-based mechanism. Absorption of UV-B induces instant monomerization of the photoreceptor and interaction with COP1, the central regulator of light signaling. Thereby this signaling cascade controlled by UVR8 mediates UV-B photomorphogenic responses securing plant acclimation and thus promotes survival in sunlight.

Interaction of the Arabidopsis UV-B-specific signaling component UVR8 with chromatin.

Arabidopsis UV RESISTANCE LOCUS8 (UVR8) is a UV-B-specific signaling component that regulates expression of a range of genes concerned with UV protection. Here, we investigate the interaction of UVR8 with chromatin. Using antibodies specific to UVR8 in chromatin immunoprecipitation (ChIP) assays with wild-type plants, we show that native UVR8 binds to chromatin in vivo. Similar experiments using an anti-GFP antibody with plants expressing a GFP-UVR8 fusion show that UVR8 associates with a relatively small region of chromatin containing the HY5 gene. UVR8 interacts with chromatin containing the promoter regions of other genes, but not with all the genes it regulates. UV-B is not required for the interaction of UVR8 with chromatin because association with several gene loci is observed in the absence of UV-B. Pull-down assays demonstrate that UVR8 associates with histones in vivo and competition experiments indicate that the interaction is preferentially with histone H2B. ChIP experiments using antibodies that recognize specific histone modifications indicate that the UV-B-stimulated transcription of some genes may be correlated with histone modification. In particular, the ELIP1 promoter showed a significant enrichment of diacetyl histone H3 (K9/K14) following UV-B exposure. These findings increase understanding of the interaction of the key UV-B-specific regulator UVR8 with chromatin.

Post-irradiation phosphorylation of structural maintenance chromosome 1 (SMC1) is independent of the Fanconi protein pathway.

PURPOSE: To confirm the sensitivity of cells from patients with Fanconi anemia (FA) to ionizing radiation, and to determine whether the phosphorylation of structural maintenance chromosome 1 (SMC1) was associated with radiosensitivity, as it is in other DNA repair disorders. METHODS AND MATERIALS: Using lymphoblastoid cell lines from FA patients before and after exposure to ionizing radiation, the colony survival assay, radioresistant DNA synthesis, and SMC1 phosphorylation were measured. FA lymphoblastoid cell lines that had been transfected with the wild-type FANC gene were used as controls. RESULTS: Cells from FA patients of six complementation groups were radiosensitive. Despite this, SMC1 phosphorylation was normal in each case; radioresistant DNA synthesis, a measure of S phase checkpoint integrity, was defective in FANCD2 lymphoblastoid cell lines and was corrected in FANCD2 + D2 cells. CONCLUSIONS: The data indicate that the FANC pathway proteins play a major role in the cellular responses to ionizing radiation, but not in SMC1 phosphorylation or in the S phase checkpoint of FANCD2-deficient cells. Thus, SMC1 activation is not a common denominator of radiosensitivity, as has been suggested by radiation responses of cells from ataxia-telangiectasia, Nijmegen breakage syndrome, or Mre11 deficiency patients.

211At-alpha-dose dependence of poly-ADP-ribosylation of human glioblastoma cells in vitro. Suitability in cancer therapy?

AIM: It was intended to test the biological response (poly-ADP-ribosylation of cellular proteins) of alpha-particles from extracellular 211At for enhanced damage to human glioblastoma cells in vitro and to discuss its suitability for potential application in therapy of high-grade gliomas. MATERIALS AND METHODS: Confluent cultures of human glioblastoma cells were exposed to different doses of alpha-radiations from homogeneously distributed extracellular 211At. Cellular poly-ADP-ribosylation of all proteins including histones was monitored since it is an indirect but sensitive indicator of chromatin damage and putative repair in both normal and malignant mammalian cells. RESULTS: A significant diminution (average 85.6%) in poly-ADP-ribosylation of total cellular proteins relative to that for non-irradiated glioblastoma cells was observed following 0.025 to 1.0 Gy alpha-radiations. In the dose range of 0.0025 to 0.01 Gy there was an increase with a maximum value of approximately 119.0% at 0.0025 Gy. Below 0.0025 Gy no change in poly-ADP-ribosylation was observed. CONCLUSIONS: Level of cellular poly-ADP-ribosylation of proteins at 0.025 to 1.0 Gy of alpha-radiation dose from 211At appears to cause enhanced damage by creating molecular conditions which are not conducive to repair of DNA damages in human glioblastoma cells in vitro. Therefore, it is assumed that clinical application of 211At at least in this dose range might enhance clinical efficacy in radiotherapy of cancer.

[The effect of gamma irradiation on the content in the thymus and liver chromatin of rats of DNA-bound proteins resistant to dissociation by phenol and sodium dodecylsulfate]. / Vliianie gamma-oblucheniia na soderzhanie v khromatine timusa i pecheni krys DNK-sviazannykh belkov, ustoichivykh k dissotsiatsii fenolom i dodetsilsul'fatom natriia.

Two groups of proteins of 50-68 kD (A) and 12-14 kD (B) are the components of DNP preparations from rat thymus and liver obtained by washing with 0.075 M NaCl-0.024 M EDTA solution and deproteinization with phenol and dodecylsulfate (SDS). Immediately after irradiation with a dose of 10 Gy, there observed an approximately 1.5-fold increase in the content of only B proteins in the rat thymus fraction precipitated upon treatment with SDS-NaCl. The acidic amino acid content of this fraction and DNP preparation obtained without treatment with SDS amounts to 25 mol%; the ratio to basic amino acids was 1.3-1.4. The comparison of the amino acid content in the above DNP preparation and the "supramolecular DNA" preparation, described in the literature, that was obtained by the same phenol deproteinization and contained about 50 mol% of acidic amino acids, indicates the presence in the "supramolecular DNA" preparation of a component that increases upon irradiation: the component consists almost completely of acidic amino acids and is eliminated completely from the DNP preparation by washing with 0.075 M NaCl-0.024 M EDTA prior to deproteinization. The amino acid composition of the protein fraction A is presented.

[Changes in the composition of DNA-bound nonhistone protein in the thymus and liver of gamma-irradiated rats]. / Izmenenie sostava DNK-sviazannogo negistonovogo belka v timuse i pecheni gamma-obluchennykh krys.

A supramolecular DNA complex (SC DNA) and DNA of a phenol nuclear matrix (PNM DNA) were extracted, by the phenol method, from rat thymus and liver 15 min following 10 Gy gamma-irradiation. The method of electrophoresis in polyacrylamide gel was used to study a composition of nonhistone proteins firmly bound to these DNA fractions. Irradiation was shown to induce the occurrence of new proteins and redistribution of proteins between SC DNA and PNN DNA of rat organs.

[Functional properties of E. coli SSB-proteins in vivo]. / Funktsional'nye svoistva SSB-belkov E. coli in vivo.

An essential function of single-stranded DNA-binding (SSB) proteins--a defense from nuclease action, determined by their first and second domains, is realized in conditions of normal cell metabolism. With the inhibition of the replicative furcula growth and total protein synthesis (imbalance state), the SSB protein function associated with the third domain and responsible for certain modifications in DNA polymerase II activity is realized. This causes DNA degeneration and increases radiosensitivity of cells.

[Effect of irradiation on the intensity of chromatin protein synthesis in the rat liver during protection with serotonin in pulsed inhibition of translation by cycloheximide]. / Vliianie oblucheniia na intensivnost' sinteza belkov khromatina v pecheni krys pri zashchite serotoninom v usloviiakh impul'snogo tormozheniia transliatsii tsiklogeksimidom.

The model of impulse translation inhibition by cycloheximide in the rat liver was used for studying the combined and separate effect of serotonin and irradiation on the intensity of synthesis of chromatin proteins (histones and nonhistone proteins). Irradiation induces a considerable inhibition of the protein synthesis intensity, while serotonin has a contrary action, that under conditions of subsequent irradiation provides an increase in the synthesis level of histones and nonhistone proteins. Possible mechanisms of the irradiation and serotonin effect on the synthesis processes of chromatin proteins in the organism are discussed.

[Synthesis and phosphorylation of histones and nonhistone proteins as affected by irradiation and serotonin in cycloheximide-synchronized hepatocytes]. / Sintez i fosforilirovanie gisotonov i negistonovykh belkov pod vliianiem oblucheniia i serotonina v sinkhronizirovannykh tsiklogeksimidom gepatotsitakh.

Phosphorylation and synthesis of histones and nonhistone proteins were studied after the inhibition of translation by sublethal cycloheximide doses. Activation of the chromatin protein phosphorylation was noted: (1) at the stage of recovery and stimulation of the protein synthesis (18-24 h), and (2) at the stage of activation of the replicative DNA synthesis (30-60 h). Phosphorylation and synthesis of the chromatin proteins depended upon the individual or combined effect of X-radiation and serotonin. The authors discuss the possible role of the chromatin protein phosphorylation in the response of the nuclear apparatus to the effect of radiation and serotonin the latter being used as a radioprotective agent.

Cross-linking of chromosomal non-histone proteins to DNA by UV radiation and some antitumor drugs.

Antibodies reacting specifically with HeLa cell chromatin can be elicited by immunization with dehistonized HeLa chromatin preparations. The nature of these chromatin-associated antigens was investigated by cross-linking with UV irradiation or by in vitro exposure of chromatin to 1-methyl-1-nitrosourea (MNU) or 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU). With the exception of 1-methyl-1-nitrosourea the described treatment of chromatin (native or dehistonized) significantly increased its immunological reactivity. Dissociation of the chromosomal proteins from DNA by concentrated salt-urea solutions essentially abolished the immunological reactivity of the residual chromatin pellets. The immunological activity was found in the supernatant protein fraction after its reconstitution with purified human placenta DNA. UV irradiation or alkylation of chromatin cross-linked the active proteins to DNA and prevented their dissociation. It is concluded that the immunologically cell-specific antigens in HeLa chromatin exist as closely associated complexes of chromosomal protein(s) with DNA.