Phosphoproteomics reveals distinct modes of Mec1/ATR signaling during DNA replication.

The Mec1/Tel1 kinases (human ATR/ATM) play numerous roles in the DNA replication stress response. Despite the multi-functionality of these kinases, studies of their in vivo action have mostly relied on a few well-established substrates. Here we employed a combined genetic-phosphoproteomic approach to monitor Mec1/Tel1 signaling in a systematic, unbiased, and quantitative manner. Unexpectedly, we find that Mec1 is highly active during normal DNA replication, at levels comparable or higher than Mec1's activation state induced by replication stress. This "replication-correlated" mode of Mec1 action requires the 9-1-1 clamp and the Dna2 lagging-strand factor and is distinguishable from Mec1's action in activating the downstream kinase Rad53. We propose that Mec1/ATR performs key functions during ongoing DNA synthesis that are distinct from their canonical checkpoint role during replication stress.

Assembly of Slx4 signaling complexes behind DNA replication forks.

Obstructions to replication fork progression, referred to collectively as DNA replication stress, challenge genome stability. In Saccharomyces cerevisiae, cells lacking RTT107 or SLX4 show genome instability and sensitivity to DNA replication stress and are defective in the completion of DNA replication during recovery from replication stress. We demonstrate that Slx4 is recruited to chromatin behind stressed replication forks, in a region that is spatially distinct from that occupied by the replication machinery. Slx4 complex formation is nucleated by Mec1 phosphorylation of histone H2A, which is recognized by the constitutive Slx4 binding partner Rtt107. Slx4 is essential for recruiting the Mec1 activator Dpb11 behind stressed replication forks, and Slx4 complexes are important for full activity of Mec1. We propose that Slx4 complexes promote robust checkpoint signaling by Mec1 by stably recruiting Dpb11 within a discrete domain behind the replication fork, during DNA replication stress.

From yeast to humans: Understanding the biology of DNA Damage Response (DDR) kinases.

The DNA Damage Response (DDR) is a complex network of biological processes that protect cells from accumulating aberrant DNA structures, thereby maintaining genomic stability and, as a consequence, preventing the development of cancer and other diseases. The DDR pathway is coordinated by a signaling cascade mediated by the PI3K-like kinases (PIKK) ATM and ATR and by their downstream kinases CHK2 and CHK1, respectively. Together, these kinases regulate several aspects of the cellular program in response to genomic stress. Much of our understanding of these kinases came from studies performed in the 1990s using yeast as a model organism. The purpose of this review is to present a historical perspective on the discovery of the DDR kinases in yeast and the importance of this model for the identification and functional understanding of their mammalian orthologues.

Investigation on solubilization protocols in the refolding of the thioredoxin TsnC from Xylella fastidiosa by high hydrostatic pressure approach.

The lack of efficient refolding methodologies must be overcome to take full advantage of the fact that bacteria express high levels of aggregated recombinant proteins. High hydrostatic pressure (HHP) impairs intermolecular hydrophobic and electrostatic interactions, dissociating aggregates, which makes HHP a useful tool to solubilize proteins for subsequent refolding. A process of refolding was set up by using as a model TsnC, a thioredoxin that catalyzes the disulfide reduction to a dithiol, a useful indication of biological activity. The inclusion bodies (IB) were dissociated at 2.4 kbar. The effect of incubation of IB suspensions at 1-800 bar, the guanidine hydrochloride concentration, the oxidized/reduced glutathione (GSH/GSSG) ratios, and the additives in the refolding buffer were analyzed. To assess the yields of fully biologically active protein obtained for each tested condition, it was crucial to analyze both the TsnC solubilization yield and its enzymatic activity. Application of 2.4 kbar to the IB suspension in the presence of 9 mM GSH, 1mM GSSG, 0.75 M guanidine hydrochloride, and 0.5M arginine with subsequent incubation at 1 bar furnished high refolding yield (81%). The experience gained in this study shall help to establish efficient HHP-based protein refolding processes for other proteins.

Opi1-mediated transcriptional modulation orchestrates genotoxic stress response in budding yeast.

In budding yeast, the transcriptional repressor Opi1 regulates phospholipid biosynthesis by repressing expression of genes containing inositol-sensitive upstream activation sequences. Upon genotoxic stress, cells activate the DNA damage response to coordinate a complex network of signaling pathways aimed at preserving genomic integrity. Here, we reveal that Opi1 is important to modulate transcription in response to genotoxic stress. We find that cells lacking Opi1 exhibit hypersensitivity to genotoxins, along with a delayed G1-to-S-phase transition and decreased gamma-H2A levels. Transcriptome analysis using RNA sequencing reveals that Opi1 plays a central role in modulating essential biological processes during methyl methanesulfonate (MMS)-associated stress, including repression of phospholipid biosynthesis and transduction of mating signaling. Moreover, Opi1 induces sulfate assimilation and amino acid metabolic processes, such as arginine and histidine biosynthesis and glycine catabolism. Furthermore, we observe increased mitochondrial DNA instability in opi1Δ cells upon MMS treatment. Notably, we show that constitutive activation of the transcription factor Ino2-Ino4 is responsible for genotoxin sensitivity in Opi1-deficient cells, and the production of inositol pyrophosphates by Kcs1 counteracts Opi1 function specifically during MMS-induced stress. Overall, our findings highlight Opi1 as a critical sensor of genotoxic stress in budding yeast, orchestrating gene expression to facilitate appropriate stress responses.

Structural and biochemical characterization of peroxiredoxin Qbeta from Xylella fastidiosa: catalytic mechanism and high reactivity.

The phytopathogenic bacterium Xylella fastidiosa is the etiological agent of various plant diseases. To survive under oxidative stress imposed by the host, microorganisms express antioxidant proteins, including cysteine-based peroxidases named peroxiredoxins. This work is a comprehensive analysis of the catalysis performed by PrxQ from X. fastidiosa (XfPrxQ) that belongs to a peroxiredoxin class still poorly characterized and previously considered as moderately reactive toward hydroperoxides. Contrary to these assumptions, our competitive kinetics studies have shown that the second-order rate constants of the peroxidase reactions of XfPrxQ with hydrogen peroxide and peroxynitrite are in the order of 10(7) and 10(6) M(-1) S(-1), respectively, which are as fast as the most efficient peroxidases. The XfPrxQ disulfides were only slightly reducible by dithiothreitol; therefore, the identification of a thioredoxin system as the probable biological reductant of XfPrxQ was a relevant finding. We also showed by site-specific mutagenesis and mass spectrometry that an intramolecular disulfide bond between Cys-47 and Cys-83 is generated during the catalytic cycle. Furthermore, we elucidated the crystal structure of XfPrxQ C47S in which Ser-47 and Cys-83 lie approximately 12.3 A apart. Therefore, significant conformational changes are required for disulfide bond formation. In fact, circular dichroism data indicated that there was a significant redox-dependent unfolding of alpha-helices, which is probably triggered by the peroxidatic cysteine oxidation. Finally, we proposed a model that takes data from this work as well data as from the literature into account.

TOPBP1Dpb11 plays a conserved role in homologous recombination DNA repair through the coordinated recruitment of 53BP1Rad9.

Genome maintenance and cancer suppression require homologous recombination (HR) DNA repair. In yeast and mammals, the scaffold protein TOPBP1Dpb11 has been implicated in HR, although its precise function and mechanism of action remain elusive. In this study, we show that yeast Dpb11 plays an antagonistic role in recombination control through regulated protein interactions. Dpb11 mediates opposing roles in DNA end resection by coordinating both the stabilization and exclusion of Rad9 from DNA lesions. The Mec1 kinase promotes the pro-resection function of Dpb11 by mediating its interaction with the Slx4 scaffold. Human TOPBP1Dpb11 engages in interactions with the anti-resection factor 53BP1 and the pro-resection factor BRCA1, suggesting that TOPBP1 also mediates opposing functions in HR control. Hyperstabilization of the 53BP1-TOPBP1 interaction enhances the recruitment of 53BP1 to nuclear foci in the S phase, resulting in impaired HR and the accumulation of chromosomal aberrations. Our results support a model in which TOPBP1Dpb11 plays a conserved role in mediating a phosphoregulated circuitry for the control of recombinational DNA repair.

Oligomerization of Cu,Zn-Superoxide Dismutase (SOD1) by Docosahexaenoic Acid and Its Hydroperoxides In Vitro: Aggregation Dependence on Fatty Acid Unsaturation and Thiols.

Docosahexaenoic acid (C22:6, n-3, DHA) is a polyunsaturated fatty acid highly enriched in the brain. This fatty acid can be easily oxidized yielding hydroperoxides as primary products. Cu, Zn-Superoxide dismutase (SOD1) aggregation is a common hallmark of Amyotrophic Lateral Sclerosis (ALS) and the molecular mechanisms behind their formation are not completely understood. Here we investigated the effect of DHA and its hydroperoxides (DHAOOH) on human SOD1 oligomerization in vitro. DHA induced the formation of high-molecular-weight (HMW) SOD1 species (>700 kDa). Aggregation was dependent on free thiols and occurred primarily with the protein in its apo-form. SOD1 incubation with DHA was accompanied by changes in protein structure leading to exposure of protein hydrophobic patches and formation of non-amyloid aggregates. Site-directed mutagenesis studies demonstrated that Cys 6 and Cys 111 in wild-type and Cys 6 in ALS-linked G93A mutant are required for aggregation. In contrast, DHAOOH did not induce HMW species formation but promoted abnormal covalent dimerization of apo-SOD1 that was resistant to SDS and thiol reductants. Overall, our data demonstrate that DHA and DHAOOH induce distinct types of apo-SOD1 oligomerization leading to the formation of HMW and low-molecular-weight species, respectively.

Structural aspects of the distinct biochemical properties of glutaredoxin 1 and glutaredoxin 2 from Saccharomyces cerevisiae.

Glutaredoxins (Grxs) are small (9-12 kDa) heat-stable proteins that are ubiquitously distributed. In Saccharomyces cerevisiae, seven Grx enzymes have been identified. Two of them (yGrx1 and yGrx2) are dithiolic, possessing a conserved Cys-Pro-Tyr-Cys motif. Here, we show that yGrx2 has a specific activity 15 times higher than that of yGrx1, although these two oxidoreductases share 64% identity and 85% similarity with respect to their amino acid sequences. Further characterization of the enzymatic activities through two-substrate kinetics analysis revealed that yGrx2 possesses a lower K(M) for glutathione and a higher turnover than yGrx1. To better comprehend these biochemical differences, the pK(a) of the N-terminal active-site cysteines (Cys27) of these two proteins and of the yGrx2-C30S mutant were determined. Since the pK(a) values of the yGrx1 and yGrx2 Cys27 residues are very similar, these parameters cannot account for the difference observed between their specific activities. Therefore, crystal structures of yGrx2 in the oxidized form and with a glutathionyl mixed disulfide were determined at resolutions of 2.05 and 1.91 A, respectively. Comparisons of yGrx2 structures with the recently determined structures of yGrx1 provided insights into their remarkable functional divergence. We hypothesize that the substitutions of Ser23 and Gln52 in yGrx1 by Ala23 and Glu52 in yGrx2 modify the capability of the active-site C-terminal cysteine to attack the mixed disulfide between the N-terminal active-site cysteine and the glutathione molecule. Mutagenesis studies supported this hypothesis. The observed structural and functional differences between yGrx1 and yGrx2 may reflect variations in substrate specificity.

Reactive cysteine in proteins: protein folding, antioxidant defense, redox signaling and more.

Cysteine plays structural roles in proteins and can also participate in electron transfer reactions, when some structural folds provide appropriated environments for stabilization of its sulfhydryl group in the anionic form, called thiolate (RS(-)). In contrast, sulfhydryl group of free cysteine has a relatively high pK(a) (8,5) and as a consequence is relatively inert for redox reaction in physiological conditions. Thiolate is considerable more powerful as nucleophilic agent than its protonated form, therefore, reactive cysteine are present mainly in its anionic form in proteins. In this review, we describe several processes in which reactive cysteine in proteins take part, showing a high degree of redox chemistry versatility.

Organic hydroperoxide resistance gene encodes a thiol-dependent peroxidase.

ohr (organic hydroperoxide resistance gene) is present in several species of bacteria, and its deletion renders cells specifically sensitive to organic peroxides. The goal of this work was to determine the biochemical function of Ohr from Xylella fastidiosa. All of the Ohr homologues possess two cysteine residues, one of them located in a VCP motif, which is also present in all of the proteins from the peroxiredoxin family. Therefore, we have investigated whether Ohr possesses thiol-dependent peroxidase activity. The ohr gene from X. fastidiosa was expressed in Escherichia coli, and the recombinant Ohr decomposed hydroperoxides in a dithiothreitol-dependent manner. Ohr was about twenty times more efficient to remove organic hydroperoxides than to remove H(2)O(2). This result is consistent with the organic hydroperoxide sensitivity of Delta ohr strains. The dependence of Ohr on thiol compounds was ascertained by glutamine synthetase protection assays. Approximately two thiol equivalents were consumed per peroxide removed indicating that Ohr catalyzes the following reaction: 2RSH + ROOH --> RSSR + ROH + H(2)O. Pretreatment of Ohr with N-ethyl maleimide and substitution of cysteine residues by serines inhibited this peroxidase activity indicating that both of the Ohr cysteines are important to the decomposition of peroxides. C125S still had a residual enzymatic activity indicating that Cys-61 is directly involved in peroxide removal. Monothiol compounds do not support the peroxidase activity of Ohr as well as thioredoxin from Saccharomyces cerevisiae and from Spirulina. Interestingly, dithiothreitol and dyhydrolipoic acid, which possess two sulfhydryl groups, do support the peroxidase activity of Ohr. Taken together our results unequivocally demonstrated that Ohr is a thiol-dependent peroxidase.

Crystallization and preliminary X-ray diffraction analysis of an oxidized state of Ohr from Xylella fastidiosa.

Xylella fastidiosa organic hydroperoxide-resistance protein (Ohr) is a dithiol-dependent peroxidase that is widely conserved in several pathogenic bacteria with high affinity for organic hydroperoxides. The protein was crystallized using the hanging-drop vapour-diffusion method in the presence of PEG 4000 as precipitant after treatment with organic peroxide (t-butyl hydroperoxide). X-ray diffraction data were collected to a maximum resolution of 1.8 A using a synchrotron-radiation source. The crystal belongs to the hexagonal space group P6(5)22, with unit-cell parameters a = b = 87.66, c = 160.28 A. The crystal structure was solved by molecular-replacement methods. The enzyme has a homodimeric quaternary structure similar to that observed for its homologue from Pseudomonas aeruginosa, but differs from the previous structure as the active-site residue Cys61 is oxidized. Structure refinement is in progress.