Architecture of the complete oxygen-sensing FixL-FixJ two-component signal transduction system

The symbiotic nitrogen-fixing bacterium Bradyrhizobium japonicum is critical to the agro-industrial production of soybean because it enables the production of high yields of soybeans with little use of nitrogenous fertilizers. The FixL and FixJ two-component system (TCS) of this bacterium ensures that nitrogen fixation is only stimulated under conditions of low oxygen. When it is not bound to oxygen, the histidine kinase FixL undergoes autophosphorylation and transfers phosphate from adenosine triphosphate (ATP) to the response regulator FixJ, which, in turn, stimulates the expression of genes required for nitrogen fixation. We purified full-length B. japonicum FixL and FixJ proteins and defined their structures individually and in complex using small-angle x-ray scattering, crystallographic, and in silico modeling techniques. Comparison of active and inactive forms of FixL suggests that intramolecular signal transduction is driven by local changes in the sensor domain and in the coiled-coil region connecting the sensor and histidine kinase domains. We also found that FixJ exhibits conformational plasticity not only in the monomeric state but also in tetrameric complexes with FixL during phosphotransfer. This structural characterization of a complete TCS contributes both a mechanistic and evolutionary understanding to TCS signal relay, specifically in the context of the control of nitrogen fixation in root nodules

Mitotic UV irradiation induces a DNA replication-licensing defect that potentiates G1 arrest response.

Cdt1 begins to accumulate in M phase and has a key role in establishing replication licensing at the end of mitosis or in early G1 phase. Treatments that damage the DNA of cells, such as UV irradiation, induce Cdt1 degradation through PCNA-dependent CRL4-Cdt2 ubiquitin ligase. How Cdt1 degradation is linked to cell cycle progression, however, remains unclear. In G1 phase, when licensing is established, UV irradiation leads to Cdt1 degradation, but has little effect on the licensing state. In M phase, however, UV irradiation does not induce Cdt1 degradation. When mitotic UV-irradiated cells were released into G1 phase, Cdt1 was degraded before licensing was established. Thus, these cells exhibited both defective licensing and G1 cell cycle arrest. The frequency of G1 arrest increased in cells expressing extra copies of Cdt2, and thus in cells in which Cdt1 degradation was enhanced, whereas the frequency of G1 arrest was reduced in cell expressing an extra copy of Cdt1. The G1 arrest response of cells irradiated in mitosis was important for cell survival by preventing the induction of apoptosis. Based on these observations, we propose that mammalian cells have a DNA replication-licensing checkpoint response to DNA damage induced during mitosis

Identifying the chloroperoxyl radical in acidified sodium chlorite solution.

The present study identified the active radical species in acidic sodium chlorite and investigated the feasibility of quantifying these species with the diethylphenylenediamine (DPD) method. Electron spin resonance (ESR) spectroscopy was used to identify the active species generated in solutions containing sodium chlorite (NaClO2). The ESR signal was directly observed in an acidified sodium chlorite (ASC) aqueous solution at room temperature. This ESR signal was very long-lived, indicating that the radical was thermodynamically stable. The ESR parameters of this signal did not coincide with previously reported values of the chlorine radical (Cl●) or chlorine dioxide radical (O = Cl●-O and O = Cl-O●). We refer to this signal as being from the chloroperoxyl radical (Cl-O-O●). Quantum chemical calculations revealed that the optimal structure of the chloroperoxyl radical is much more thermodynamically stable than that of the chlorine dioxide radical. The UV-visible spectrum of the chloroperoxyl radical showed maximum absorbance at 354 nm. This absorbance had a linear relationship with the chloroperoxyl radical ESR signal intensity. Quantifying the free chlorine concentration by the DPD method also revealed a linear relationship with the maximum absorbance at 354 nm, which in turn showed a linear relationship with the chloroperoxyl radical ESR signal intensity. These linear relationships suggest that the DPD method can quantify chloroperoxyl radicals, which this study considers to be the active species in ASC aqueous solution

Ectopic expression of Cdt1 lowers the G1 arrest response.

<p>A. Extra copy of Cdt1 expressing cells have more MCM2-7 on the chromatin. Control (293) and Cdt1-3NLSmyc expressing HEK293 cells (Cdt1) were arrested in M phase and released with (M-UV) or without [(-)UV] UV irradiation (60 J/m²). Each cell line was collected at the indicated time-points for preparation of whole cell extracts (WCE) and an insoluble fraction. B. Flow cytometry analysis of Cdt1-3NLSmyc expressing cells. Cells treated as in A were collected 24 h after release for flow cytometry. To block cells in mitosis, nocodazole was added at the 10 h time-point. C. Cdt1-3NLSmyc expressing cells show a higher apoptotic response. Cells treated as in A were cultured for the indicated days, and collected to monitor the cleavage of caspase 3 (Cas-3) by immunoblotting. Cleaved caspase-3 levels in UV-irradiated cells were normalized to those of non-irradiated cells for each cell line.</p

Cells released after UV irradiation in M phase show a stronger G1 arrest phenotype than cells UV-irradiated in G1 phase.

<p>A. Cdt1 degradation and chromatin association of proteins. HeLa cells synchronized in M phase were released (-), UV-irradiated at 40 J/m², and released 10 min later (M-UV) or UV-irradiated after release at 3 h (G1-UV), and collected at the indicated time-points for the preparation of whole cell extracts (WCE) and an insoluble fraction. The indicated proteins were examined by Western blotting. B. Flow cytometry analysis of cells UV-irradiated during M phase or in G1 phase. Cells treated as in A were collected at 16 h after release for flow cytometry. C. Levels of CPD. Cells UV-irradiated in M phase or G1 phase at 3 h after release were measured for CPD levels by dot blotting. D. Levels of checkpoint activation. Cells UV-irradiated in M phase or in G1 phase at 3 h after release and collected at the indicated time-points for measurement of the P-S296 levels. E. Sensitivity of cells to UV irradiation in M phase or G1 phase. Cells UV-irradiated in M phase or G1 phase at 3 h after release were cultured for 2 weeks, and colony formation was measured. Triplicate experiments were performed. The colony numbers were normalized, set to 100% for UV(-) cells.</p

UV irradiation induces Cdt1 degradation in G1 phase, but not in mitosis.

<p>A. Mcm6 remains associated with chromatin after UV irradiation in G1 phase. Half of the dishes of mitotic HeLa cells, prepared as described in Materials and Methods, were released into G1 phase and collected at the indicated time-points and the other half of the dishes of cells were UV-irradiated at 3 h (+3) or 5 h (+5) after release and collected at 6 h. Insoluble fractions were prepared after centrifuging the cell lysates and blotted with the indicated antibodies. RCC1 was used as a control. B. Mitotic HeLa cells (M phase) were incubated without any treatment (-), irradiated with UV (50 J/m²) and incubated (+), or incubated in the presence of MMS (1 mM) (+) for the indicated time (h). Cells were collected, and whole cell extracts were made and blotted with the indicated antibodies. Asynchronously growing cells (asy) treated similarly (+) or not (-) and collected 1 h later were included as a control. Closed arrow-heads indicate the hyperphosphorylated forms of Cdt1, and open arrowhead indicates the fast migrating form of G1 phase. * indicates bands not specific for Cdt1. C. Asynchronously growing HeLa cells were treated with nocodazole at 40 ng/ml (+) for 6 or 9 h or not (-), irradiated with UV (50 J/m²) and incubated for 1 h. Whole cell extracts were prepared and blotted with the indicated antibodies. PCNA was used as a loading control. D. Mitotic HeLa cell cultures (M phase) were UV-irradiated (50 J/m²) (+), treated with MMS (1 mM) (+) or not (-) (-1 h), and incubated for1 h, then cells were washed out of nocodazole (and MMS) for release into G1 phase for 0, 2, and 4 h. Whole cell extracts were made and blotted with the indicated antibodies. Cyclin B was used to monitor the exit from mitosis (Cyc.B). Asynchronously growing cells (asy) treated similarly (+) or not (-) and collected 1 h later were included as a control. E. Control or PIP-box mutated Cdt1 (PIP-Cdt1:A6-Cdt1-3NLSmyc) expressing HEK293 cells were synchronized in M phase, UV-irradiated (40 J/m²) (+) or not (-), and released into G1 phase. Cells were collected at time 0 and 5 h, whole cell extracts were prepared and blotted with the indicated antibodies. Endogenous Cdt1 (end-.) and PIP-box mutated Cdt1 (PIP-) were indicated.</p

Ectopic expression of Cdt2 enhances the G1 arrest response.

<p>A. Cdt1 degradation and MCM2-7 loading in control HEK293 cells and Cdt2-FLAG expressing HEK293 cells. Control (293) and Cdt2-FLAG expressing HEK293 cells (Cdt2) were arrested in M phase and released with or without UV irradiation (40 J/m²). Cells were collected at the indicated time-points to prepare whole cell extracts (WCE) and an insoluble fraction. Note that Cdt2 (both endogenous (end.) and FLAG-tagged (Cdt2-FLAG)) were hyperphosphorylated in M phase[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120553#pone.0120553.ref025" target="_blank">25</a>]. B. Flow cytometry analysis of Cdt2-FLAG expressing cells. Cells treated as in A were collected 24 h after release for flow cytometry. To block cells in mitosis, nocodazole was added at the 10 h time-point. Frequency of G1 phase cells was shown (%).</p

UV-irradiated M phase-cells show decreased MCM 2–7 loading and G1 arrest after release: HeLa cells.

<p>A. Scheme of synchronization and sample preparation. DT block: double thymidine block. B and C. HeLa cells synchronized in M phase were released without UV irradiation [(-)UV], or UV-irradiated at 25 J/m² or 50 J/m² [(+)UV] and released 10 min later. Cells were collected at the indicated time-points for flow cytometry (B) and for preparation of whole cell extracts (WCE) and insoluble fractions for immunoblotting (C). D. Immunofluorescent analysis of MCM2-7 loading. Cells released without UV irradiation [(-)UV] or after UV irradiation in M phase (M-UV) or in G1 phase at 3 h after release (G1-UV) were pre-extracted and fixed at 8 h and stained for Mcm3. Mcm3 signals for each cell were measured and the signal intensity distribution profile was plotted.</p

UV-irradiated M-phase cells show decreased MCM 2–7 loading and G1 arrest after release: HEK293 cells and U2OS cells.

<p>A. HEK293 cells synchronized in M phase were released without UV irradiation [(-)UV], or UV-irradiated with 50 J/m² [(+)UV] and released 10 min later. Cells were collected and treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120553#pone.0120553.g002" target="_blank">Fig. 2B and 2C</a>. WCE, whole cell extract. B. U2OS cells UV-irradiated in M phase and released. U2OS cells synchronized in M phase were released or UV-irradiated at 50 J/m² or 100 J/m² and released 10 min later. Cells were collected and treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120553#pone.0120553.g002" target="_blank">Fig. 2B and 2C</a>.</p

Mismatch repair proteins recruited to ultraviolet light-damaged sites lead to degradation of licensing factor Cdt1 in the G1 phase

<p>Cdt1 is rapidly degraded by CRL4^Cdt2 E3 ubiquitin ligase after UV (UV) irradiation. Previous reports revealed that the nucleotide excision repair (NER) pathway is responsible for the rapid Cdt1-proteolysis. Here, we show that mismatch repair (MMR) proteins are also involved in the degradation of Cdt1 after UV irradiation in the G1 phase. First, compared with the rapid (within ∼15 min) degradation of Cdt1 in normal fibroblasts, Cdt1 remained stable for ∼30 min in NER-deficient XP-A cells, but was degraded within ∼60 min. The delayed degradation was also dependent on PCNA and CRL4^Cdt2. The MMR proteins Msh2 and Msh6 were recruited to the UV-damaged sites of XP-A cells in the G1 phase. Depletion of these factors with small interfering RNAs prevented Cdt1 degradation in XP-A cells. Similar to the findings in XP-A cells, depletion of XPA delayed Cdt1 degradation in normal fibroblasts and U2OS cells, and co-depletion of Msh6 further prevented Cdt1 degradation. Furthermore, depletion of Msh6 alone delayed Cdt1 degradation in both cell types. When Cdt1 degradation was attenuated by high Cdt1 expression, repair synthesis at the damaged sites was inhibited. Our findings demonstrate that UV irradiation induces multiple repair pathways that activate CRL4^Cdt2 to degrade its target proteins in the G1 phase of the cell cycle, leading to efficient repair of DNA damage.</p