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Electron micrograph of metastatic thyroid carcinoma cell. For further information see article e00426. Image provided by Jianru Xiao
AUTHOR CONTRIBUTION
Research Article| December 05 2016
The DnaE polymerase from Deinococcus radiodurans features RecA-dependent DNA polymerase activity
Lorenzo Randi;
Lorenzo Randi
*Department of Pharmacy and Biotechnology, University of Bologna, Viale Risorgimento 4, 40136 Bologna, Italy
Alessandro Perrone;
Alessandro Perrone
Mirko Maturi;
Mirko Maturi
Fabrizio Dal Piaz;
Fabrizio Dal Piaz
†Department of Medicine, University of Salerno, Via Giovanni Paolo II, 132, 84084 Fisciano SA, Italy
Michela Camerani;
Michela Camerani
Alejandro Hochkoeppler
Alejandro Hochkoeppler 1
‡CSGI, University of Firenze, Via della Lastruccia 3, 50019 Sesto Fiorentino (FI), Italy
1To whom correspondence should be addressed (email a.hochkoeppler@unibo.it).
https://doi.org/10.1042/BSR20160364
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Lorenzo Randi, Alessandro Perrone, Mirko Maturi, Fabrizio Dal Piaz, Michela Camerani, Alejandro Hochkoeppler; The DnaE polymerase from Deinococcus radiodurans features RecA-dependent DNA polymerase activity. Biosci Rep 1 December 2016; 36 (6): e00419. doi: https://doi.org/10.1042/BSR20160364
We report in the present study on the catalytic properties of the Deinococcus radiodurans DNA polymerase III α subunit (αDr). The αDr enzyme was overexpressed in Escherichia coli, both in soluble form and as inclusion bodies. When purified from soluble protein extracts, αDr was found to be tightly associated with E. coli RNA polymerase, from which αDr could not be dissociated. On the contrary, when refolded from inclusion bodies, αDr was devoid of E. coli RNA polymerase and was purified to homogeneity. When assayed with different DNA substrates, αDr featured slower DNA extension rates when compared with the corresponding enzyme from E. coli (E. coli DNA Pol III, αEc), unless under high ionic strength conditions or in the presence of manganese. Further assays were performed using a ssDNA and a dsDNA, whose recombination yields a DNA substrate. Surprisingly, αDr was found to be incapable of recombination-dependent DNA polymerase activity, whereas αEc was competent in this action. However, in the presence of the RecA recombinase, αDr was able to efficiently extend the DNA substrate produced by recombination. Upon comparing the rates of RecA-dependent and RecA-independent DNA polymerase activities, we detected a significant activation of αDr by the recombinase. Conversely, the activity of αEc was found maximal under non-recombination conditions. Overall, our observations indicate a sharp contrast between the catalytic actions of αDr and αEc, with αDr more performing under recombination conditions, and αEc preferring DNA substrates whose extension does not require recombination events.
Deinococcus radiodurans, DnaE polymerase, DNA polymerase III, Escherichia coli, RecA, α subunit
DNA, Chromosomes & Chromosomal Structure, Enzymology
DNA polymerases (DNA Pols) are peculiar enzymes, featuring a conserved multi-domain molecular architecture. In particular, the conformation of DNA Pols resembles an open right-hand and contains three main domains, denoted by thumb, palm and fingers [1]. The thumb domain tightly associates with the double-stranded portion of the DNA substrate, whereas the 3′-OH end of the primer and the ssDNA template are allocated in the palm and in the fingers domain respectively [2]. The subsequent binding of a deoxynucleoside-triphosphate (dNTP) triggers the movement of the fingers towards the palm, poising the active site to proficient catalysis, i.e. the nucleophilic attack by the 3′-OH of the primer to the α-phosphate of the dNTP [3]. Besides this primary DNA extension activity, the complex task of genome replication demands to DNA Pols the exertion of quite a number of secondary essential functions. Among these functions, high fidelity and processive replication, as well as proper co-ordination of leading and lagging DNA strand extension, are accomplished by DNA Pol holoenzymes composed of different subunits, the assembly of which determines the actual enzyme activities [2]. Escherichia coli does express five distinct DNA Pols, with DNA Pol III responsible for genome replication [4–6], DNA Pol I essential for removing the multiple primers necessary for lagging strand replication [7,8] and DNA Pols II, IV and V [9–12], eventually engaged in the replication of damaged DNA. E. coli DNA Pol III (αEc) is composed of a catalytic core, a DnaX complex and a β-clamp. The catalytic core does contain the α, ε and θ subunits [13] respectively featuring 5′–3′ polymerase [14], 3′–5′ exonuclease (proofreading) [15] and ε-stabilizing activity [16,17]. Notably, DNA Pol III α subunit, which is coded by the dnaE gene, is the only essential replicase in E. coli. The DnaX complex contains the τ, δ, δ', χ and Ψ subunits [18], according to a stoichiometry τ3δδ'χψ [19]. This complex catalyses the loading of the β-clamp on to DNA, and interacts with the core α subunit. The β-clamp is a homodimer, and confers to the holoenzyme high processivity by tethering the polymerase to the DNA substrate [20,21]. In addition, the progression of a replication fork is assisted by the association of DNA Pol III with DNA helicase and primase [22,23]. Interestingly, it was shown that β-clamp, helicase and primase are competent in interacting with E. coli DNA Pols II, IV and V [24], whose action is assisted by the RecA recombinase [25–27]. More recently, it was also elegantly demonstrated that RecA activates replisomes containing DNA Pol II, Pol IV or Pol V [28], and inhibits DNA Pol III holoenzyme [28]. In addition, it was observed that the overexpression of Pol IV and RecA slows down the progression of Pol III replication forks [29], suggesting that RecA inhibits the action of Pol III holoenzyme in E. coli.
Deinococcus radiodurans is a Gram-positive bacterium, belonging to the Deinococcales order, whose members feature outstanding radioresistance [30]. D. radiodurans was indeed first isolated from canned meat samples, exposed to γ-rays in order to obtain a sterile, and hence stable, food supply [31]. Contrary to the vast majority of prokaryotes, D. radiodurans cells are polyploid, with the actual ploidy number being affected by growth phase [32] and culture medium [33]. Each genome copy consists of two chromosomes (containing 2.6 and 0.4 Mbp) and two plasmids, featuring 177×103 and 45.7×103 bp respectively [34]. Three different DNA Pols were identified in D. radiodurans, i.e. DNA Pol I [35], Pol III [36] and Pol X [37], among which DNA Pol III α subunit (DnaE) is essential for viability [36,38]. Rather astonishingly, cells of D. radiodurans exposed to 14 kGy, and containing fragmented chromosomes, are able to reconstruct their genomes within 6–7 h after radiation exposure [30]. Although polyploidy is an obvious requisite for this genome reconstruction competence, D. radiodurans does also feature additional and peculiar biochemical properties responsible for genome integrity maintenance. In particular, when considering that ionizing radiations induce severe oxidative stress, it was realized that the radiation-resistance of D. radiodurans is mainly due to biochemical factors preserving the proteome of this bacterium from oxidation damages [30]. Nevertheless, the capability of D. radiodurans to reconstruct its highly fragmented genome does also imply an outstanding DNA repair competence. To accomplish this task, D. radiodurans first depends on RecJ, which is essential for viability [39] and features 5′–3′ exonuclease activity. The action of this enzyme produces 3′ overhangs at the expense of chromosomal/plasmid fragments, triggering the RecFOR-mediated loading of RecA on to DNA. Subsequently, the recombinase activity of RecA and the DNA polymerase activity of DnaE catalyse the recombination of overlapping homologous fragments and the extension of the recombinant molecules respectively. This mechanism, denominated extensive synthesis-dependent strand annealing (ESDSA), does progressively increase the molecular mass of DNA fragments, whose number decreases in parallel. Besides the impairment of ESDSA, both recA and dnaE mutations confer interesting phenotypes to D. radiodurans: (i) ΔrecA strains feature poor viability and extreme radiation sensitivity [39–41]; (ii) under physiological conditions (no irradiation), recA mutants do replicate DNA at slower rates when compared with wild-type [36]; (iii) temperature-sensitive dnaE mutants feature lower radiation-resistance than strains defective in DNA polymerase I (PolA) [35,36]. In particular, the poor DNA replication efficiency of unirradiated RecA− strains suggests a role for this recombinase in DnaE-catalysed replication.
Both DNA Pol I and DNA Pol X from D. radiodurans were previously purified, and their catalytic performances were investigated [35,42–45]. On the contrary, the α subunit (DnaE) of D. radiodurans DNA Pol III (αDr) has never been isolated to homogeneity in vitro. In the present study, we report on the overexpression and purification of this enzyme, along with the characterization of its catalytic properties at the expense of different DNA substrates, in the presence or in the absence of RecA. Further, we used the well-known α subunit (DnaE) of E. coli DNA Pol III (here denominated αEc) as a reference polymerase, and we report on the comparison of these two enzymes, whose action is essential for their respective organisms.
Bacterial strains, plasmids and growth media
Escherichia coli BW25993 [genotype: Δ(araD-araB)567, rph-1, Δ(rhaD-rhaB)568, hsdR514] was obtained by Mary Berlyn (Escherichia coli Genetic Stock Center, CGSC, Yale University), and E. coli TOP10 [genotype: F−mcrA Δ(mrr-hsdRMS-mcrBC) ϕ80lacZΔM15 ΔlacX74 recA1 araD139 Δ(ara-leu)7697 galU galK rpsL endA1 nupG] was purchased from Invitrogen. Both strains were grown in LB medium (10, 10 and 5 g/l respectively, of Tryptone, NaCl and Yeast Extract), supplemented, when appropriate, with 100 μg/ml of ampicillin. The plasmid pBAD-α1160, containing an insert coding for the full-length α subunit of E. coli DNA Polymerase III (αEc), was previously described [46], and will be indicated here as pBAD-αEc. The gene coding for the full-length α subunit of Deinococcus radiodurans DNA Polymerase III (αDr) was optimized for E. coli codon usage and synthesized by GenScript. The synthetic gene was inserted into the pBAD-HisB plasmid, using the NcoI and PstI sites at 5′ and 3′ ends respectively, yielding pBAD-αDr. This vector was used to transform both E. coli strains BW25993 and TOP10, the transformants were isolated and purified on LB Petri dishes containing 100 μg/ml of ampicillin, and then stored at −20°C in LB–glycerol (15%, v/v) medium. Proteins overexpression was triggered in LB medium by the addition of 1 mM arabinose.
Proteins overexpression
The overexpression of αEc was performed as recently described [47]. The overexpression of soluble αDr was attempted using both E. coli strains TOP10 and BW25993. In E. coli TOP10, the target protein was expressed at low concentrations, irrespective of temperature for growth and induction length. On the contrary, the BW25993 strain did perform much better, and was used throughout to overexpress soluble αDr. In particular, single colonies of E. coli BW25993/pBAD-αDr were picked from LB–ampicillin Petri dishes, transferred to LB–ampicillin liquid medium and pre-cultured at 37°C for 8 h. The pre-cultures were then diluted (1:500) in fresh LB medium, grown for 15 h at 37°C, and the cultures were finally induced at 37°C for 3 h. Cells were collected by centrifugation (10000 × g, 20 min, 4°C), and the pellets were stored at −20°C until used.
To overexpress αDr in the form of inclusion bodies, single colonies of E. coli TOP10/pBAD-αDr were pre-cultured in LB medium for 15 h at 37°C. The pre-cultures were then diluted (1:500) in fresh LB medium, grown for 8 h, and finally induced for 15 h at 37°C. After harvest, the cell pellets were stored at −20°C.
Extraction of soluble proteins
To isolate soluble protein extracts, cells were resuspended in Buffer A, consisting of 50 mM Tris/HCl (pH 8), 50 mM NaCl, 1 mM EDTA, 2.5 mM DTT, and gently homogenized with a cold glass potter. The cell suspension was then supplemented with 1 mM PMSF and subjected to 7 cycles of sonication at 6 W. Each sonication cycle consisted of 15 s of impulse, followed by 15 s of cooling interval, for a total time of 2 min. The extracts accordingly obtained were centrifuged (10000 × g, 20 min, 4°C), the pellets were discarded, and the supernatant was immediately used to purify soluble proteins.
Solubilization of proteins from inclusion bodies
E. coli TOP10/pBAD-αDr cells were disrupted as described for the extraction of soluble proteins. After sonication, the extract was centrifuged at 4000 × g (20 min, 4°C), and the pellet (containing the inclusion bodies) was washed twice with 200 mM Tris/HCl (pH 8), 500 mM NaCl, 1 mM DTT, 0.1% (v/v) Triton-X-100. After the second wash, the pellet was harvested and stored at −20°C until used. To solubilize proteins, inclusion bodies were resuspended in 200 mM Tris/HCl (pH 8), 500 mM NaCl, 5 mM β-mercaptoethanol, 6 M urea, using 100 ml of buffer per g (wet weight) of sample. After incubation at room temperature for 3 h under magnetic stirring, the solution was centrifuged at 16000 × g (20 min, 20°C), and the pellet was discarded, yielding a supernatant containing 50 mg of proteins per g of inclusion bodies. To refold proteins, the supernatant was subjected to four dialysis steps, against: (i) 200 mM Tris/HCl (pH 8), 500 mM NaCl, 5 mM β-mercaptoethanol, 2 mM MgCl2, 4 M urea; (ii) 100 mM Tris/HCl, 500 mM NaCl, 5 mM β-mercaptoethanol, 2 mM MgCl2, 2 M urea; (iii) 50 mM Tris/HCl, 200 mM NaCl, 5 mM β-mercaptoethanol, 2 mM MgCl2, 1 M urea and (iv) 50 mM Tris/HCl, 50 mM NaCl, 5 mM β-mercaptoethanol, 2 mM MgCl2, 10% (v/v) glycerol. The dialysis tube contained 30 ml of sample per litre of external buffer.
Purification of soluble proteins
The α subunit of E. coli DNA Pol III was purified as previously described [47], and the same procedure was used to isolate homogeneous αDr. Briefly, the soluble protein extracts were subjected to anion-exchange chromatography (Q-Sepharose FF, 50 ml column) in Buffer A, applying a linear 50–600 mM NaCl gradient. The best fractions, according to SDS/PAGE analysis, were pooled, desalted, concentrated, supplemented with 20% (v/v) glycerol, 5 mM MgCl2 and loaded on to a Cibacron Blue column (15 ml). After extensive washing of the column with Buffer B (Buffer A containing 20% glycerol and 5 mM MgCl2), αDr was eluted with the same buffer containing 1 M NaCl. The eluted peak was desalted and loaded on to a HiTrap Heparin column (5 ml), to which a linear 50–600 mM gradient of NaCl was applied. The best fractions were reconditioned to 50 mM NaCl with Buffer A containing 20% glycerol, and they were finally subjected to a second anion-exchange chromatography, using a HiTrap Q column (5 ml). The best fractions, containing partially purified αDr, were pooled and stored at −20°C until used.
Purification of αDr from inclusion bodies
The refolded sample was loaded on to a Cibacron Blue column (15 ml), and after a washing step (six column volumes), αDr was eluted with Buffer B containing 1 M NaCl. After dialysis against Buffer A containing 20% glycerol, the sample was loaded on to a HiTrap Heparin column (5 ml) and subjected to a linear 50–600 mM NaCl gradient. The best fractions were pooled and stored at −20°C until used.
Elution of proteins from acrylamide gels, trypsin digestion and separation of peptides were performed as previously described [17]. The resulting peptides were analysed by LC-MS/MS using an Orbitrap XL instrument (Thermo Fisher Scientific) equipped with a nano-ESI source coupled with a nano-Acquity capillary UPLC (Waters). Briefly, peptides were separated with a capillary BEH C18 column (0.075×100 mm, 1.7 μM, Waters) using aqueous 0.1% formic acid (A) and CH3CN containing 0.1% formic acid (B) as mobile phases. Peptides were eluted by means of a linear gradient from 5 to 50% of B in 90 min, at a 300 nl/min flow rate. Mass spectra were acquired over an m/z range from 400 to 1800. To achieve protein identification, MS and MS/MS data underwent Mascot Search Engine software analysis to interrogate the National Center for Biotechnology Information nonredundant (NCBInr) protein database. Parameters sets were: trypsin cleavage; carbamidomethylation of cysteines as a fixed modification and methionine oxidation as a variable modification; a maximum of two missed cleavages; false discovery rate, calculated by searching the decoy database, 0.05.
Enzyme assays
Type XV activated calf-thymus DNA was obtained from Sigma–Aldrich. E. coli RecA recombinase and dNTPs mixture (dATP, dGTP, dTTP and dCTP, 10 mM each) were purchased from NEB. Oligonucleotides, to be annealed and used as DNA substrates, were synthesized by GenScript. DNA polymerase activity was determined according to our previously described enzyme-coupled assay, which was designed for the continuous spectrophotometric detection of PPi/phosphate [48]. Briefly, reactions were assayed in 100 mM Tris/HCl (pH 8), 5 mM MgCl2, 0.25 mM inosine, 100 μM dNTPs and 10, 50 and 500 m-units/ml of inorganic pyrophosphatase (PPase), purine nucleoside phosphorylase (PNPase) and xanthine oxidase (XOD) respectively. The uric acid generated by XOD was detected at 293 nm, and phosphate concentrations were calculated assuming a molar absorption coefficient for uric acid equal to 12600 M−1 · cm−1 [49]. The ATPase activity of RecA was determined detecting both the Pi and the ADP generated. To continuously determine the release of ADP, the reaction mixture contained 100 mM Tris/HCl (pH 8), 10 mM MgCl2, 20 mM KCl, 0.5 mM phosphoenolpyruvate (PEP), 1.5 unit/ml of pyruvate kinase (PK), 0.25 mM β-NADH and 6 units/ml of lactate dehydrogenase (LDH). By this means, the ATP consumed by RecA is regenerated from ADP and PEP, which is converted to pyruvate by PK; finally, pyruvate is reduced, in the presence of β-NADH, to lactate. Reactions were monitored at 340 nm, and the molar absorption coefficient of β-NADH was assumed equal to 6200 M−1 · cm−1.
The lag time before steady-state intermediate concentrations is reached, as we observed in the present study, is well-known for enzyme coupled assays [50]. Activities were determined by considering the interval time, subsequent to the lag, during which zero-order kinetics was detected.
All the assays were performed using a Cary 300 UV–VIS spectrophotometer. Protein concentration was determined according to Bradford [51], using BSA as standard.
Overexpression of soluble D. radiodurans DnaE polymerase
We first attempted the overexpression of D. radiodurans αDr in soluble form. To this aim, we transformed E. coli TOP10 with pBAD-αDr, and we searched for growth and induction conditions corresponding to overexpression yields compatible with the purification of the target protein. However, using E. coli TOP10/pBAD-αDr we were not able to obtain satisfactory amounts of soluble αDr (results not shown). On the contrary, when E. coli BW25993/pBAD-αDr was grown at 37°C and induced for 3 h at the same temperature, high overexpression of soluble αDr was observed (Supplementary Figure S1A). Soluble protein extracts were accordingly prepared from this bacterial host, and the purification of αDr was pursued by four consecutive chromatographic steps (see Materials and Methods). By this means, we significantly purified αDr (Supplementary Figure S1B and Table 1), but we could not dissociate from the DNA polymerase an accompanying contaminant, which was identified by MS as E. coli RNA polymerase (Tables 2 and 3). Considering that the concentration of this partially purified αDr cannot be quantified, we used this enzyme preparation to perform qualitative assays, the aim of which was to confirm what we observed using refolded αDr (see below).
Peptides of αDr identified by MS
Identification by MS of partially purified D. radiodurans DnaE polymerase.
Peptide .
Observed Mr (Da) .
Theoretical Mr (Da) .
Sequence .
240–254 1597.8406 1597.8311 SDATAHETLLAIQTK.A
359–370 1201.5936 1201.5938 SLAELGEADAAR
525–539 1847.9742 1847.9669 ITNLDPLEFELLFER
671–681 1315.6844 1315.6806 EELTNLVPVMR
696–704 972.5504 972.5491 SVEDIGLIK
1035–1044 1055.4694 1055.4672 SGAFDAFGER
1089–1099 1236.6036 1236.5986 SSIAPYSDLER
1105–1123 2118.0931 2118.0858 EALGLYISGHPLEQHEGLR
Peptides of E. coli RNA polymerase β subunit identified by MS
Identification by MS of E. coli RNA polymerase β subunit, the presence of which was detected as contaminant of D. radiodurans DnaE polymerase, overexpressed in soluble form and subjected to four consecutive purification steps.
38–54 1968.9212 1968.9217
89–97 962.5186 962.5185
119–143 2858.3134 2858.3103
144–151 950.5662 950.5661
997–1007 1331.6616 1331.6609
1008–1022 1847.8900 1847.8901
1148–1156 978.4770 978.4771
Peptides of E. coli RNA polymerase α subunit identified by MS
Identification by MS of E. coli RNA polymerase α subunit, the presence of which was detected as contaminant of D. radiodurans DnaE polymerase, overexpressed in soluble form and subjected to four consecutive purification steps.
1–12 1407.7180 1407.7180
92–104 1455.8317 1455.8297
Refolding and purification of D. radiodurans DnaE polymerase from inclusion bodies
To pursue the isolation of highly purified αDr, devoid of E. coli RNA polymerase, we decided to attempt the refolding of the enzyme from inclusion bodies. The induction for 15 h at 37°C of E. coli TOP10/pBAD-αDr yielded 0.9 g (wet weight) of inclusion bodies per litre of culture. Upon solubilization with urea (6M), and four consecutive dialysis steps performed as described by Brown et al. [52] for yeast DNA polymerase δ, we finally obtained 27 mg of soluble proteins per g of inclusion bodies, among which αDr was highly abundant. This refolded sample was then subjected to Cibacron Blue and HiTrap Heparin affinity chromatography (Supplementary Figure S2). With both chromatographic supports, a consistent amount of αDr was lost with the flow-through, most likely containing improperly refolded enzyme. From 27 mg of dialysed proteins, we were able to recover 3.6 mg of refolded and purified αDr.
DNA polymerase activity of refolded αDr and αEc
We compared the catalytic performances of refolded αDr with those of the well-known αEc. To this aim, we first assayed the DNA polymerase activity of both enzymes at the expense of a DNA 40mer template annealed to a 15mer primer, the extension of which exclusively depends on dTTP (Figure 1). The extension of DNA was monitored by our previously described enzyme-coupled assay (PPX assay, [48]), which relies on (1): (i) inorganic PPase, the action of which releases Pi at the expense of the PPi generated by a DNA Pol; (ii) PNPase, which in the presence of inosine and Pi, catalyses the release of ribose-1-phosphate and hypoxanthine and (iii) XOD, finally converting hypoxanthine to uric acid, which is conveniently detectable at 293 nm [49]. Under these conditions, the activity of αDr was found to be approximately 90 times lower when compared with that of the E. coli enzyme (0.19 and 17 nM/s respectively, Figures 2A and 2B, and Supplementary Figures S3A and S3B). However, it was also observed that αDr is significantly stimulated by ionic strength, its DNA polymerase activity being approximately 4-fold higher in the presence of 200 mM NaCl or KCl (Figure 2A, and Supplementary Figure S3A). On the contrary, αEc was dramatically inhibited, i.e. 30-fold, by 200 mM KCl (Figure 2B, and Supplementary Figure S3B). Therefore, under these conditions αDr performs slightly better than its E. coli homologue. Moreover, when calf-thymus activated DNA was used as substrate, and all the four dNTPs were present in reaction mixtures, comparable activities were observed in the presence of αDr or αEc (0.25 and 0.3 nM/s respectively, Figure 2C, and Supplementary Figure S3C). It was previously reported that D. radiodurans DNA Pols I and X are positively affected by Mn2+ [42,44]. Accordingly, we assayed αDr in the presence of manganese, the 40mer dsDNA and dTTP. Surprisingly, a strong activation of αDr was observed, yielding an activity equal to 5.5 nM/s (Supplementary Figure S3D), whereas a small effect, if any, was detected in the presence of αEc under the same conditions (results not shown).
Reaction paths leading to the generation of uric acid at the expense of Pi and inosine (upper row), and to the oxidation of β-NADH from ADP and PEP (lower row). Although the determination of uric acid can be used to assay both DNA polymerase and RecA activity, the evaluation of β-NADH oxidation is useful to specifically assay RecA in the presence of concomitant DNA polymerase activity.
Cartoon and sequences of the different DNAs used in the present study.
DNA polymerase activities detected with αDr and αEc at the expense of different DNAs. All the activity values are expressed as the increase in PPi concentration (nM) per unit time (s)
(A) Elongation rate of 40mer dsDNA by 34 nM αDr under conditions of low ionic strength (Control, dark red bar) or in the presence of 200 mM NaCl (blue bar) or KCl (pink bar). (B) Extension rate of 40mer dsDNA by 33 nM αEc under conditions of low ionic strength (Control, dark red bar) or in the presence of 200 mM NaCl (blue bar). (C) Rate of activated calf thymus DNA elongation catalysed by 34 nM αDr (green bar) or by 33 nM αEc (orange bar).
A continuous assay for recombination-dependent DNA polymerase activity
We thought it of interest to assay the activity of αDr and αEc under conditions requiring recombination to generate a dsDNA substrate. Accordingly, we designed three oligodeoxynucleotides, two of which to be annealed into a linear 48mer dsDNA, and the third to be used as 60mer ssDNA in the assay (Figure 1). By this means, DNA polymerase activity should not be observed at the expense of dTTP, unless a previous recombination step between the 48mer dsDNA and the 60mer ssDNA occurs, generating a primer contiguous to a polyA template (Figure 1). As a first test, we assayed the binding efficiency of E. coli RecA to the DNAs to be recombined. To this aim, we took advantage of our PPX assay, useful to detect the RecA ATPase activity triggered by the association of this enzyme to DNA. In agreement with previous observations, we detected ATP hydrolysis coupled to the binding of RecA to ssDNA, whereas dsDNA was found to be a poor substrate (Figure 3A). We then assayed the RecA-dependence of αEc polymerase activity, using both 48mer dsDNA and 60mer ssDNA, in the presence of different dNTPs. As shown in Figures 3(B) and 3(C), only in the presence of dTTP does αEc feature RecA-dependent polymerase activity, whereas the addition of dGTP to reaction mixtures triggers the extension of DNA independently of the recombinase. Therefore, to determine recombination-dependent DNA polymerase activity, we decided to use reaction mixtures containing dsDNA, ssDNA, dTTP, RecA and ATP. In addition, we designed an assay for the simultaneous detection of the ATPase activity of RecA and the DNA polymerase activity of αEc or αDr. Our PPX assay does not indeed discriminate between DNA Pol activity and the ATPase activity of RecA. However, we thought that an additional reaction path could be coupled to RecA, i.e. the regeneration of ATP catalysed by PK at the expense of PEP, and the reduction of pyruvate to lactate by LDH, in the presence of β-NADH (1). Using this strategy, the activity of RecA could be monitored by quantifying the consumption of β-NADH, and DNA Pol activity would be identified estimating the amount of Pi linked to PPi hydrolysis by PPase. It should also be mentioned that the two molar absorption coefficients of β-NADH and uric acid are 6200 and 12600 M−1 · cm−1 respectively. As shown in Figure 4(A), when the absorption spectrum of a solution containing ssDNA, RecA, ATP, inosine, PNPase, XOD, PEP, β-NADH, PK and LDH was recorded as a function of time, the band of uric acid at 293 nm did progressively increase, and the band of β-NADH did concomitantly decrease, indicating the release by RecA of Pi from ATP. Moreover, if the absorbance values at 293 and at 340 nm are both considered as a function of time, the kinetics of RecA activity can be reliably quantified (Figure 4B). As a further test of our continuous assay for recombination-dependent DNA polymerase activity, denominated here as PPX-PL assay, we recorded absorption spectra of solutions containing a complete reaction mixture, i.e. dsDNA, ssDNA, dTTP, αEC, inosine, PPase, PNPase, XOD, RecA, ATP, PEP, β-NADH, PK and LDH. Under these conditions, the increase in absorption at 293 nm was more than twice the absorbance decrease at 340 nm, indicating the presence of DNA Pol activity triggered by the recombination of dsDNA and ssDNA (Figure 4C). Moreover, when the first minutes of the reaction are analysed in detail, it can be observed that recombination occurs first, and then DNA Pol activity takes place (Figure 4D).
ATPase activity of E. coli RecA and assays of αEc DNA extension activity
(A) The release of Pi from ATP catalysed by 1 μM RecA was assayed in the presence of 40mer ssDNA (green dots) or at the expense of 40mer dsDNA (blue dots). (B) Assay of the Pi released by 1 μM RecA from 1 mM ATP and of the PPi generated by 33 nM αEc in the presence of 48mer dsDNA and 60mer ssDNA, 1 μM each (see Figure 1). Reaction mixtures contained inosine, PPase, PNPase and XOD as coupling enzymes. Reactions were started with dGTP and dTTP (100 μM each, green dots), dGTP (blue dots) or dTTP (red dots). (C) Assay of the PPi generated by 33 nM αEc in the presence of 48mer dsDNA and 60mer ssDNA, 1 μM each, in the absence of RecA. Other conditions are described in (B).
Assays of E. coli RecA ATPase activity and of αEc recombination-dependent DNA elongation
(A) Difference absorption spectra recorded 5, 15, 25 and 35 min (pink, red, blue and green lines respectively) after addition of 1 μM RecA to a reaction mixture containing 1 mM ATP, 1 μM 60mer ssDNA, inosine, PEP, β-NADH, PNPase, XOD, PK and LDH. A cuvette containing a reaction mixture devoid of RecA was used as reference. (B) Absorbance differences as a function of time detected at 293 nm (diamonds) and 340 nm (circles) under the conditions of Figure 1(A). (C) Difference absorption spectra recorded during the first 10 min after addition of 33 nM αEc to a reaction mixture containing 1 μM RecA, 1 mM ATP, 48mer dsDNA and 60mer ssDNA (1 μM each), 100 μM dTTP, inosine, PEP, β-NADH, PPase, PNPase, XOD, PK and LDH. A cuvette containing a reaction mixture devoid of RecA and αEc was used as reference. (D) Kinetics of PPi release exerted by αEc during recombination-dependent DNA elongation (diamonds), and time course of RecA ATPase activity (circles), under the conditions of (C). The RecA ATPase activity was determined by quantifying the coupled oxidation of β-NADH, and the release of PPi by αEc was calculated via the coupled generation of uric acid, to the total concentration of which was subtracted the concentration generated by the action of RecA.
Recombination-dependent polymerase activity of αEc and αDr
The recombination-dependent polymerase activity of αDr was tested with the PPX-PL assay, by recording absorption spectra every 10 min after the reaction was started by the addition of RecA and αDr. As shown in Figure 5(A), αDr did efficiently catalyse the extension of the dsDNA generated by E. coli RecA. From Figure 5(A), it is indeed clearly visible that the amplitude of the uric acid band does promptly exceed more than twice the amplitude of the negative band related to β-NADH consumption. The corresponding Δabsorbance values at 293 and 340 nm, as a function of time, were used to calculate the concentration of total Pi and β-NADH oxidized (Figure 5B). From the total Pi concentration, we then subtracted the fraction generated by RecA (corresponding to the decrease in β-NADH concentration), and we accordingly obtained the concentration of PPi released by αDr (Figure 5B). Surprisingly, αDr did perform DNA extension according to a zero-order kinetics for the first 60 min of reaction time (Figure 5C), and the magnitude of this activity was significantly higher than the DNA Pol activity observed at the expense of a dsDNA not requiring recombination to be extended (cf. Figures 2A and 6A). On the contrary, when αEc was assayed under the same conditions (Figures 5D–5F), the activity of this DNA Pol was clearly much lower than the DNA extension activity observed in the absence of RecA (cf. Figures 2B and 6A). Moreover, a clear lag was observed before the DNA Pol activity of αEc entered zero-order kinetics (Figure 5F), and this kinetic regime was maintained for approximately 20 min only (Figure 5F). Overall, these observations suggest that αDr is stimulated by E. coli RecA, and that under the conditions of the PPX-PL assay αDr is more processive than αEc. Accordingly, we wanted to test the DNA Pol activity of both αDr and αEc under conditions requiring recombination, but in the absence of both RecA and ATP (and therefore devoid of PK, PEP, LDH and β-NADH). By this means, we wanted to assay the intrinsic recombination activity, if at all, of αDr and αEc. Surprisingly again, although αEc was clearly able to catalyse the recombination and extension of DNA, albeit at a moderate rate, αDr was incapable to perform these tasks, most likely because of its inefficiency in recombination activity (Figure 6B, and Supplementary Figure S4).
Recombination-dependent DNA extension activity of αDr and αEc assayed in the presence of RecA
(A) Difference absorption spectra recorded every 10 min after addition of 34 nM αDr to a reaction mixture containing 1 μM RecA, 1 mM ATP, 48mer dsDNA and 60mer ssDNA (1 μM each), 100 μM dTTP, inosine, PEP, β-NADH, PPase, PNPase, XOD, PK and LDH. A cuvette containing a reaction mixture devoid of RecA and αDr was used as reference. (B) Kinetics of total Pi released by the action of RecA and αDr (filled circles), of PPi generated by αDr (diamonds), and of β-NADH oxidation linked to RecA action (empty circles). (C) Detail of the kinetics reported in (B), and representing the PPi generated by the recombination-dependent DNA extension activity of αDr. (D–F) Recombination-dependent DNA polymerase activity of 33 nM αEc, as determined under the same conditions of (A)–(C).
Recombinase action of RecA and recombination-dependent DNA extension activity of αDr and αEc assayed in the presence or in the absence of RecA
The activity values of RecA are expressed as the increase in phosphate concentration (nM), generated at the expense of ATP, per unit time (s). The DNA extension activity values are expressed as the increase in PPi concentration (nM) per unit time (s). (A) ATPase activity of RecA detected in the presence of αDr or αEc (green and blue bar respectively). The DNA polymerase activity of αDr and αEc are reported with dark green and dark blue bars respectively. Assay conditions are described in Figure 5. (B) Recombination-dependent DNA extension activity of αDr (green bar) and αEc (blue bar) assayed in the absence of RecA. The corresponding kinetics is reported in Supplementary Figure S4.
Recombination-dependent polymerase activity of αDr overexpressed in soluble form
To further investigate the RecA-dependence of αDr polymerase activity, we performed recombination-dependent DNA extension assays using partially purified αDr overexpressed in soluble form. When the polymerase activity of αDr was tested using the PPX-PL assay (in the presence of RecA), we observed a significant extension, at the expense of dTTP, of the dsDNA generated by RecA (Figure 7). On the contrary, no DNA polymerase activity was detected when αDr was added to a reaction mixture devoid of RecA, and containing the two DNAs to be recombined and dTTP (Supplementary Figure S5). These observations qualitatively agree with those obtained using refolded αDr isolated and purified from inclusion bodies.
Recombination-dependent DNA extension activity of αDr (25 μg of P/ml) overexpressed in soluble form assayed in the presence of RecA
(A) Difference absorption spectra recorded every 10 min after addition of αDr to a reaction mixture containing 1 μM RecA, 1 mM ATP, 48mer dsDNA and 60mer ssDNA (1 μM each), 100 μM dTTP, inosine, PEP, β-NADH, PPase, PNPase, XOD, PK and LDH. A cuvette containing a reaction mixture devoid of RecA and αDr was used as reference. (B) Kinetics of total Pi released by the action of RecA and αDr (filled circles), of PPi generated by αDr (diamonds), and of β-NADH oxidation linked to RecA action (empty circles). (C) Detail of the kinetics reported in (B), and representing the PPi generated by the recombination-dependent DNA extension activity of αDr (filled diamonds). As a comparison, the recombination-dependent DNA polymerase activity of 34 nM refolded αDr is also reported (empty diamonds).
The purification of the αDr is described in the present study for the first time. Rather surprisingly, when overexpressed in soluble form, αDr featured a strong binding to the RNA polymerase of the host, i.e. E. coli. Accordingly, we were able to partially purify native αDr associated with E. coli RNA Pol, and we observed that the dissociation of this complex does not occur under different conditions, e.g. gel filtration or high concentrations of DTT (results not shown). Whether or not αDr does also feature high affinity for D. radiodurans RNA Pol remains to be ascertained. In the present study, we used the αDr–RNA Pol complex for qualitative activity assays only, designed to avoid any interference by RNA Pol on the observed activities. It should be indeed noted that we performed DNA Pol activity assays relying on dTTP as the unique nucleotide substrate. To obtain homogeneous αDr free of RNA Pol, we overexpressed αDr as inclusion bodies, and we were successful in the refolding and purification of the target protein.
When compared with E. coli DNA polymerase III α subunit (αEc), αDr showed quite different kinetic properties. Under standard conditions (5 mM MgCl2, 100 μM dTTP, low ionic strength) and in the presence of 1 μM 40mer template DNA, αDr was indeed found to catalyse DNA extension with much lower efficiency than αEc. Interestingly, when the ionic strength of the assay mixture was increased, the catalytic action of αDr and αEc was found to increase and decrease respectively. Moreover, the addition of Mn2+ to the reaction milieu triggered a strong increase in αDr activity, and did correspond to a modest, if at all, effect towards the action of αEc. It is interesting to note that γ-irradiation induces a general increase in cellular ionic strength, and that the accumulation of Mn2+ increases the radioresistance of D. radiodurans [53]. Therefore, our observations suggest that the activity of αDr is regulated by the conditions arising in cells upon exposure to γ-rays. In particular, the positive response of αDr to Mn2+ reported here agrees with previous studies concerning D. radiodurans Pol I and Pol X. It was indeed demonstrated that Mn2+ is essential for the activity of D. radiodurans Pol X [37], and is required to confer competence in DNA lesion bypass to DNA Pol I [42]. Accordingly, the DNA Pols of D. radiodurans share a strong dependence on manganese of their activities, suggesting a nice correlation with the high content of manganese that is usually detected in post-irradiated cells of this organism. However, it should also be considered that other DNA Pols, isolated from radiation-sensitive organisms, depend on Mn2+ for their activity, e.g. the human ιDNA Pol [54].
When considering that the ESDSA pathway of genome reconstitution is strictly dependent on αDr, one might expect that this DNA polymerase features intrinsic recombinase activity. However, αDr was found to be incapable of recombination-dependent DNA polymerase activity, independently of the strategy used to overexpress αDr in E. coli (Supplementary Figures S4 and S5). On the contrary, this activity was exclusively detected in the presence of both RecA and αDr. This is in sharp contrast with the catalytic properties of αEc, which was found competent in recombination-dependent DNA polymerase activity in the absence of RecA (Supplementary Figures S4 and S5). It should also be noted that the activity of αDr in the presence of RecA significantly exceeds the extension rate observed with a recombination-independent DNA substrate (cf. Figures 2A and 6A), suggesting an activation of αDr by RecA. This evidence agrees with the finding that genome replication in D. radiodurans RecA− strains is slower when compared with that observed in the wild-type counterpart [36]. Conversely, deletion of RecA does not significantly affect the growth rate of E. coli [55]. Moreover, it was demonstrated that the overexpression of recA decreases the rate of replication fork progression in E. coli [29], presumably by inhibiting αEc replisomes [28].
We observed that the rate and the extent of ATP hydrolysis differ if αDr or αEc were present in RecA-dependent DNA extension assays. During the first 60–70 min after reactions started, we did indeed ascertain that: (i) when αDr was present, 5 μM dTTP was incorporated into DNA, and the hydrolysis of 10–15 μM ATP was concomitantly detected (Figure 8A); (ii) when αEc did catalyse the extension of DNA at the expense of 4–5 μM dTTP, RecA did hydrolyse 20–30 μM ATP (Figure 8B). These findings suggest that αDr does assist RecA in the recombination reaction, lowering the amount of ATP necessary to sustain strand exchange and, hence, DNA extension. Interestingly, the energetics of RecA-mediated recombination was finely dissected by a series of elegant studies. First, it was demonstrated that the rate of ATP hydrolysis triggered by the association of RecA with ssDNA slows down upon initiation of strand exchange with dsDNA [56]. Further, it was recognized that: (i) the binding of ATP or of ATP analogues converts RecA inactive filaments to an active state [57]; (ii) ATP hydrolysis and recombinational branch migration are concerted reactions [58]. Moreover, it was also shown that ATP hydrolysis induces a conformational transition of RecA C-terminus, and that this transition corresponds to the conversion of inactive RecA filaments into their active counterpart [59,60]. Finally, different states of RecA filaments were identified [61]. In the absence of ATP, RecA is in the O state and is competent in binding ssDNA [61]. When ATP is present, the recombinase switches to the A (activated) state, and the binding of ssDNA triggers ATP hydrolysis. The A state can be further characterized in two sub-states, denoted Ac (A closed) and Ao (A open), to indicate incompetence and competence in pairing reactions respectively [61]. The transition from the Ac to the Ao state was found to depend on Mg2+ concentration [62]. In the presence of a pairing substrate, RecA switches from the Ao to the P state, and this transition is paralleled by a decrease (approximately 30%) of the ATP hydrolysis rate [61]. According to these observations, we propose that αDr favours the transition from the Ao to the P state of RecA, therefore limiting the amount of ATP necessary to accomplish recombination. In particular, in the presence of αDr the ATP consumed per nucleotide incorporated in DNA was equal to approximately 2, whereas in the presence of αEc this ratio is more than double (Figure 8).
Kinetics of RecA-dependent DNA extension catalysed by αDr and αEc
(A) Detail of Figure 5(B) showing the extension of DNA (diamonds) and the hydrolysis of ATP (filled circles) catalysed by αDr and RecA respectively, during the first 80 min after reactions started. (B) Detail of Figure 5(E) showing the extension of DNA (diamonds) and the hydrolysis of ATP (filled circles) catalysed by αEc and RecA respectively, during the first 80 min after reactions started. The concentration of PPi as a function of time (corresponding to DNA polymerase activity) was calculated by subtracting the observed total Pi concentration to the fraction generated by RecA, and dividing the residuals by 2.
Overall, we have shown in the present study that some of the catalytic properties of αDr are in sharp contrast with those of αEc: (i) αDr is stimulated by high ionic strength, whereas αEc is strongly inhibited under the same conditions; (ii) similarly to other DNA Pols of D. radiodurans, αDr is activated by Mn2+, whereas αEc action is not significantly affected by manganese; (iii) contrary to αEc, αDr is activated by RecA, in the absence of which αDr is not able to catalyse recombination-dependent DNA extension. Remarkably, the actions of E. coli and D. radiodurans RecA proteins were reported to differ substantially [63,64]. Although E. coli RecA does indeed prefer ssDNA as substrate (as also shown here, see Figure 3), the D. radiodurans recombinase was reported to bind more efficiently to dsDNA. Accordingly, future work will be devoted to investigate the catalytic action of αDr in recombination-dependent DNA polymerase activity assays performed in the presence of D. radiodurans RecA protein.
Lorenzo Randi and Michela Camerani purified αDr in soluble form. Alessandro Perrone and Mirko Maturi purified αEc and αDr from inclusion bodies. Fabrizio Dal Piaz did perform mass-spectrometry experiments. Lorenzo Randi, Michela Camerani, Alessandro Perrone, Mirko Maturi and Alejandro Hochkoeppler assayed the activity of αDr and αEc. Alejandro Hochkoeppler wrote the manuscript.
DNA Pols
DNA polymerases
dNTP
deoxynucleoside-triphosphate
αDr
Deinococcus radiodurans DNA polymerase III αsubunit
αEc
Escherichia coli DNA Pol III
ESDSA
extensive synthesis-dependent strand annealing
phosphoenolpyruvate
pyruvate kinase
PNPase
purine nucleoside phosphorylase
PPase
inorganic pyrophosphatase
PPase-PNPase-XOD enzyme coupled assay
PPX-PL
PPase-PNPase-XOD-PK-LDH enzyme coupled assay
XOD
xanthine oxidase
Steitz
Function and structure relationships in DNA polymerases
Annu. Rev. Biochem.
(pg.
https://doi.org/10.1146/annurev.bi.63.070194.004021
DNA replicases from a bacterial perspective
https://doi.org/10.1146/annurev-biochem-061208-091655
Bermek
Grindley
Distinct roles of the active-site Mg2+ ligands, Asp882 and Asp705, of DNA polymerase I (Klenow fragment) during the prechemistry conformational transitions
Gefter
M.L.
Kornberg
Barnoux
Analysis of DNA polymerases II and III in mutants of Escherichia coli thermosensitive for DNA synthesis
Proc. Natl. Acad. Sci. U.S.A.
Nüsslein
Function of DNA polymerase III in DNA replication
Nat. New Biol.
Deoxyribonucleic acid synthesis in cell-free extracts. IV. Purification and catalytic properties of deoxyribonucleic acid polymerase III
De Lucia
Isolation of an E. coli strain with a mutation affecting DNA polymerase
https://doi.org/10.1038/2241164a0
Method for determining whether a gene of Escherichia coli is essential: application to the polA gene
J. Bacteriol.
DNA polymerase II
Maenhaut-Michel
Sofuni
Nohmi
Ohmori
Multiple pathways for SOS-induced mutagenesis in Escherichia coli: an overexpression of dinB/dinP results in strongly enhancing mutagenesis in the absence of any exogenous treatment to damage DNA
Reuven
Tomer
Livneh
The mutagenesis proteins UmuD' and UmuC prevent lethal frameshifts while increasing base substitution mutations
Mol. Cell
Eritja
Biochemical basis of SOS-induced mutagenesis in Escherichia coli: reconstitution of in vitro lesion bypass dependent on the UmuD'2C mutagenic complex and RecA protein
DNA polymerase III of Escherichia coli. Purification and identification of subunits
Horiuchi
The polymerase subunit of DNA polymerase III of Escherichia coli. I. Amplification of the dnaE gene product and polymerase activity of the α subunit
Scheuermann
Echols
A separate editing exonuclease for DNA replication: the ε subunit of Escherichia coli DNA polymerase III holoenzyme
Taft-Benz
Schaaper
The θ subunit of Escherichia coli DNA polymerase III: a role in stabilizing the ε proofreading subunit
https://doi.org/10.1128/JB.186.9.2774-2780.2004
Vincelli
Bressanin
Dal Piaz
Hochkoeppler
Stabilization of the Escherichia coli DNA polymerase III ε subunit by the θ subunit favors in vivo assembly of the Pol III catalytic core
Arch. Biochem. Biophys.
https://doi.org/10.1016/j.abb.2012.04.013
Pritchard
Dallmann
H.G.
In vivo assembly of the τ-complex of the DNA polymerase III holoenzyme expressed from a five-gene artificial operon. Cleavage of the τ-complex to form a mixed γ-τ-complex by the OmpT protease.
https://doi.org/10.1074/jbc.271.17.10291
McInerney
Characterization of a triple DNA polymerase replisome
https://doi.org/10.1016/j.molcel.2007.06.019
P.M.
Sakakibara
The dnaN gene codes for the β subunit of DNA polymerase III holoenzyme of Escherichia coli
Kuriyan
Three-dimensional structure of the β subunit of E. coli DNA polymerase III holoenzyme: a sliding DNA clamp
https://doi.org/10.1016/0092-8674(92)90445-I
Ueda
McMacken
dnaB protein of Escherichia coli. Purification and role in the replication of ϕ;X174 DNA
Primase, the dnaG protein of Escherichia coli. An enzyme which starts DNA chains
Indiani
L.D.
Yurieva
Translesion DNA polymerases remodel the replisome and alter the speed of the replicative helicase
R.W.
Roles of the Escherichia coli RecA protein and the global SOS response in effecting DNA polymerase selection in vivo
https://doi.org/10.1128/JB.187.22.7607-7618.2005
Mallik
Popodi
Interactions and localization of Escherichia coli error-prone DNA polymerase IV after DNA damage
https://doi.org/10.1128/JB.00101-15
Jaszczur
T.M.
A RecA protein surface required for activation of DNA polymerase V
PLoS Genet.
pg.
https://doi.org/10.1371/journal.pgen.1005066
RecA acts as a switch to regulate polymerase occupancy in a moving replication fork
Furukohri
Recombinase and translesion DNA polymerase decrease the speed of replication fork progression during the DNA damage response in Escherichia coli cells
https://doi.org/10.1093/nar/gkv044
Radman
Oxidative stress resistance in Deinococcus radiodurans
Microbiol. Mol. Biol. Rev.
https://doi.org/10.1128/MMBR.00015-10
H.C.
Studies on a radio-resistant Micrococcus. I. Isolation, morphology, cultural characteristics, and resistance to gamma radiation
Food Technol.
Multiplicity of genome equivalents in the radiation-resistant bacterium Micrococcus radiodurans
Harsojo, Kitayama
Genome multiplicity and radiation resistance in Micrococcus radiodurans
J. Biochem.
Gwinn
W.C.
Genome sequence of the radioresistant bacterium Deinococcus radiodurans R1
Ouyang
Minton
Identification, sequencing, and targeted mutagenesis of a DNA polymerase gene required for the extreme radioresistance of Deinococcus radiodurans
Recombination and replication in DNA repair of heavily irradiated Deinococcus radiodurans
Lecointe
Shevelev
I.V.
Bailone
Hübscher
Involvement of an X family DNA polymerase in double-stranded break repair in the radioresistant organism Deinococcus radiodurans
Mol. Microbiol.
Mattingly
Shimmin
Isolation and some properties of temperature-sensitive mutants of Micrococcus radiodurans defective in DNA synthesis
J. Gen. Microbiol.
Bentchikou
A major role of the RecFOR pathway in DNA double-strand-break repair through ESDSA in Deinococcus radiodurans
Sensitization to radiation by loss of recombination ability in a temperature-sensitive DNA mutant of Micrococcus radiodurans held at its restrictive temperature
In vivo damage and recA-dependent repair of plasmid and chromosomal DNA in the radiation-resistant bacterium Deinococcus radiodurans
Lesion bypass activity of DNA polymerase A from the extremely radioresistant organism Deinococcus radiodurans
https://doi.org/10.1074/jbc.M611404200
Jolivet
DNA polymerase X from Deinococcus radiodurans possesses a structure-modulated 3′→5′ exonuclease activity involved in radioresistance
Khairnar
H.S.
DNA polymerase X from Deinococcus radiodurans implicated in bacterial tolerance to DNA damage is characterized as a short patch base excision repair polymerase
https://doi.org/10.1099/mic.0.029223-0
Leulliot
Cladière
van Tilbeurgh
The family X DNA polymerase from Deinococcus radiodurans adopts a non-standard extended conformation
Cianchetta
Proteolysis of the proofreading subunit controls the assembly of Escherichia coli DNA polymerase III catalytic core
https://doi.org/10.1016/j.bbapap.2009.07.011
Montón Silva
Lapenta
Perrone
Simultaneous ternary extension of DNA catalyzed by a trimeric replicase assembled in vivo
Guillén Suárez
Continuous enzyme-coupled assay of phosphate- or pyrophosphate-releasing enzymes
BioTechniques
Bergmeyer
H.U.
Uric acid: UV-assay with uricase
Methoden der Enzymatischen Analyse
Weinheim (D)
Verlag Chemie
W.R.
Kinetic analysis of coupled enzyme assays
https://doi.org/10.1021/bi00835a014
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding
Anal. Biochem.
Purification and characterization of the Saccharomyces cerevisiae DNA polymerase δ overproduced in Escherichia coli
Gaidamakova
Matrosova
V.Y.
Vasilenko
Zhai
Omelchenko
Kostandarithes
Makarova
Accumulation of Mn(II) in Deinococcus radiodurans facilitates gamma-radiation resistance
Increased catalytic activity and altered fidelity of human DNA polymerase ι in the presence of manganese
Meza Mora
Barrón
Ramírez
Engineering Escherichia coli to increase plasmid DNA production in high cell-density cultivations in batch mode
Microb. Cell Fact.
Schutte
Homology-dependent changes in adenosine 5′-triphosphate hydrolysis during recA protein promoted strand exchange: evidence for long paranemic complexes
Egelman
Structural data suggest that the active and inactive forms of the RecA filament are not simply interconvertible
J. Mol. Biol.
https://doi.org/10.1016/0022-2836(92)90702-L
Evidence for the coupling of ATP hydrolysis to the final (extension) phase of RecA protein-mediated DNA strand exchange
https://doi.org/10.1074/jbc.271.10.5725
Domain structure and dynamics in the helical filaments formed by RecA and Rad51 on DNA
https://doi.org/10.1073/pnas.111005398
VanLoock
ATP-mediated conformational changes in the RecA filament
A DNA pairing-enhanced conformation of bacterial RecA proteins
Lusetti
Magnesium ion-dependent activation of the RecA protein involves the C terminus
A.K.
E.A.
Jambura
RecA protein from the extremely radioresistant bacterium Deinococcus radiodurans: expression, purification, and characterization
The RecA proteins of Deinococcus radiodurans and Escherichia coli promote DNA strand exchange via inverse pathways
This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution Licence 4.0 (CC BY).
Supplementary Figures- pdf file


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After a long period of absence, we come back with some novelties, the main one is that we have a new Consul . Theresa will share the organisations of monthly dinners, which is a real pleasure.
This time we have chosen an excellent place for celebrating the reunion with old and new Friends.
The restaurant, Torres y Garcia, is industrial in style with hints of rustic appeal, it specializes in regional ingredients prepared in an open wood oven. The roomy restaurant's eclectic dining areas inspire contrasting ambient vibes. There are stark walls and brick accents, frescoes and modern art centering around an imperial table, and a wall of gourmet goods.
As a novelty, every guest will choose his/her own menu and will pay accordingly.
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"In the age of the cloud, there's no room for souvenirs," is the message of Ford Racing's showroom island design at the American Express Global Business Travel trade show. A bold, colorful display incorporating the company's biophilic branding and design elements. The brand is printed on a white background and pops up on the lighted counter. The Ford Racing showroom uses the space smartly by displaying products inside and outside the stand. The brand logo appears on a clear background and its distinctive features are displayed on the board. The exhibits are also designed to use lighting techniques, including the use of color and light. In addition to the American Express exhibit, the TelMeNow exhibit uses bold color and lighting techniques. The TelMeNow brand is displayed against a white background and the counter glows from below.
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redpajama_set_name: RedPajamaCommonCrawl


text:
Q: C# LINQ Return Generic List Below are two classes I have sampled.
Without using tuple;
  I would like to send queries directly from the first list to the second list of results.
The part that fails in the encoding appears as convert operations.
I thank you for your time and reply.
        static void Main(string[] args)
    {

        List<liste> personel = new List<liste>{

            new liste { PersonId = 1, Name = "Burak", Surname = "Şenyurt", City = "İstanbul", Salary = 890 },
            new liste { PersonId = 2, Name = "Maykıl", Surname = "Cordın", City = "Chicago", Salary = 930 },
            new liste { PersonId = 3, Name = "Şakiyıl", Surname = "Oniyıl", City = "Los Angles", Salary = 986 },
            new liste { PersonId = 4, Name = "Ümit", Surname = "Oniyıl", City = "Los Angles", Salary = 1035 },
            new liste { PersonId = 5, Name = "Mehmet", Surname = "Zaferoğlu", City = "Los Angles", Salary = 1265 },
            new liste { PersonId = 6, Name = "Hasan", Surname = "Orkun", City = "Los Angles", Salary = 1435 },
            new liste { PersonId = 7, Name = "Raşit", Surname = "Mesut", City = "Los Angles", Salary = 1469 },
            new liste { PersonId = 8, Name = "Hamdi", Surname = "Tanpınar", City = "Los Angles", Salary = 1535 },
            new liste { PersonId = 9, Name = "Şevki", Surname = "Çapkın", City = "Los Angles", Salary = 1636 },
            new liste { PersonId = 10, Name = "Özhun", Surname = "Bozkurt", City = "Los Angles", Salary = 1839 }
        };
        double resAVG = personel.Select(x => x.Salary).Average();
        List<Sonuc> reportResult = GetReport(personel,resAVG);
    }

Static Method
        public static List<Sonuc> GetReport(List<liste> listePersonel , double resAVG)
    {
        List<Sonuc> result = (from e in listePersonel
                      where e.Salary >= resAVG
                      orderby e.Salary descending
                    //select new Tuple<string, string, double>(e.Name, e.Surname, e.Salary)).ToList<Tuple<string, string, double>>();
        select new List<Sonuc>(e.Name, e.Surname, e.Salary)).ToList<Sonuc>(result.ToList());

        return result;
    }

General Class
   public class liste
{
    public int          PersonId        { get; set; }
    public string       Name            { get; set; }
    public string       Surname         { get; set; }
    public string       City            { get; set; }
    public double       Salary          { get; set; }

    public override string ToString()
    {
        return $"PersonId : {PersonId}\t\tName , Surname {Name} , {Surname}\t\t\tSalary : {Salary}";
    }
}

Result Class
    public class Sonuc
{
    public string       Name            { get; set; }
    public string       Surname         { get; set; }
    public double       Salary          { get; set; }

    public Sonuc(string Name , string Surname, double Salary)
    {
        this.Name = Name;
        this.Surname = Surname;
        this.Salary = Salary;
    }

    public override string ToString()
    {
        return $"Name, SurName : {this.Name} ,   {this.Surname}\t\t\tSalary : {this.Salary}";
    }
}

A: You're trying to construct an instance of List<T> by passing it a string, a string, and a double. List<T> does not have a constructor which takes these parameters. Also, you can't use result before you've assigned it.
Instead, you should project each item in listePersonel to a single instance of Sounc, then take that enumerable to a List<Sounc>.
public static List<Sonuc> GetReport(List<liste> listePersonel , double resAVG)
{
    List<Sonuc> result = (from e in listePersonel
                  where e.Salary >= resAVG
                  orderby e.Salary descending
                  select new Sonuc(e.Name, e.Surname, e.Salary)).ToList();

    return result;
}

redpajama_set_name: RedPajamaStackExchange


You need more aluminum foil. On your head.
Maybe we would hate you a little less if you put a little more effort into saying "aluminium".
Have you seen the size of my head? You'd need to hire a crane.
Just get a garbage can and make a helmet out of it.
First of all, I should probably empty it of all of Sir2u's awful comebacks.
On paper, the prognosis is good.
I do not suggest a globalist approach. I suggest regionalism. Regional trade blocs are emerging all across the world, EU, AU, ASEAN, and so on. These do not suffer, to the same extent global government would suffer, from the problem of perceived legitimacy. Regional government has a natural interest in promoting internal markets - which promote human welfare, in turn necessary to slow the growth of human population. Further, because nations tend to trade most with their immediate and close neighbours - the cost of regulations applied across a region like the EU, with 28 countries, are mitigated, because a cost that applies equally to direct economic competitors is not a competitive disadvantage. So regional government can afford to have higher regulatory standards, and the market is too large to be threatened by big companies wanting a race to the bottom for profit.
Don't worry, there won't be 11 billion people by 2100.
Ok, I'm not sure what this is all about, but it looks anti-Trump so it must be (a) true, and (b) significant.
It's true but a website definition and not the statutory definition. It now seems to match the statutory definition used in the Violence Against Women Act. That Act expired during the government shutdown but appears to have been extended again until 15 February. Whether there's a policy change related to the change in definition isn't clear. Reason to pay attention for now.
Fact check of Donald Trump's speech.
Trump has apparently thrown another Twitter hissy fit in response to an SNL impression by Alec Baldwin, asking how media outlets get away with such mockery, and implying that there should be "retribution." Other than perhaps Richard Nixon, has there in recent memory been a POTUS with such clearly anti-democratic tendencies?
It's bad enough that Trump praises dictators (or quasi-dictators) such as Putin, Erdogan, Kim Jong Un, and Duterte, but he himself has already floated the idea of using FCC powers to revoke the broadcast licenses of media outlets which displease him, even, apparently, when this displeasure results from the time-honored tradition of an SNL impersonation, the rite of passage for each and every POTUS over the past 30 years or so. He has also invoked yet again his labeling of the press as "the enemy of the people," a page from the dictator textbook. What a thin-skinned, cruel, vain man-child.
"I don't care. I believe Putin."
So did anyone else watch the Cohen testimony?
Some of it. I cannot stomach the republicans.
They made it difficult to watch through grandstanding, misplaced moralizing, and incoherent attacks, and overall hypocrisy. I don't understand how some of them were elected. Higgins was an absolute moron, as was Meadows, Jim Jordan is just a boot-licking dipshit.
The bits of it I saw were a bit insane. What the hell has happened to the american democracy? Are these kinds of elected representatives the result of extreme gerrymandering? Or is there some sort of collective insanity going on?
From what I read in the newspapers it was really entertaining.
I really wonder what will happen after Trump. It brings to my mind questions like a) What damage has Trump done to the GOP? b) What is the counter-response to Trump when the political pendulum swings back? Above all, c) What happens to American political discourse? Is the discourse salvageable or does the discourse continue breaking apart to two different camps that basically don't talk to each other, stay in their own worlds and result in a situation where to reach normal political compromises, that a democracy needs to function, becomes impossible.
So. Manafort got a slap on the wrist because the judge identifies better with the perpetrator than the victim(s). Class justice.
EDIT: How is it even relevant Manafort doesn't stand trial for collusion? That's like doing a search in a house because of an investigation in theft and finding drugs. It's not even remotely relevant to the sentence.
What would be interesting to see if Ellis has dealt with tax fraud before and whether he thought the guidelines were excessive then as well and that he reached very low sentences too.
This is a major failing of the justice system. Perhaps we need a corollary to the principle that one be tried before a jury of one's peers, that one should not be sentenced by one's peer. It would be possible to co-opt an appropriate panel of shop-workers and taxi drivers to deal with errant bankers, politicians, and lawyers.
Just based on the number of convictions of Trump's associates and the 17 investigations of Trump himself, I think we can safely say this is an unusually corrupt and inept administration.
We went from Obama to this. If the amplitude of the pendulum swing continues to escalate, we'll have Gandhi for president in 2020.
Trump's presidency is proof that my fellow U.S. American citizens are very stupid. We elected a stupid racist, homophobic, sexist idiot.
Here is what bothers me. I am a medical professional, and my colleagues are medical professionals. It is amazing that my co-workers can exhibit the type of logic necessary to save and/or maintain the safety of human life, yet value the ideas of Trump daily. It's almost like when discussing politics they warp in time and lose all sense of reality.
completely agree, I get narcissistic, egomaniacs exist, the fact that otherwise normal people voted and after all this, continue to support one for POTUS amazes me.
The majority of voters didn't vote for him, though. The mechanics of the electoral college allow for the minority to impose their electoral will on the majority under certain circumstances. Democratic strongholds tend to be clustered in cities (low area/high population), whereas Republican strongholds tend to be more diffuse and rural (large area/low population).
If you look at a color-coded electoral map of the U.S., it is a sea of red with a few small blue islands sprinkled in. This phenomenon, inter alia, allows Republican candidates to prevail in some national elections, even when they lose the popular vote.
But it's marginal isn't it? So slightly less than half of US voters supported a complete tosspot. Hurrah for the good sense of US voters? I'd really like if complete tosspots lost by a country mile.
We needn't postulate a false dilemma between "US voters are smart" and "US voters are stupid." I'd also like it if tosspots (assuming that's something bad - I don't speak UK) lost by a country mile, but it's also important to keep in mind how the electoral college system works, and how the person elected to POTUS doesn't always reflect the will of (most of) the people.
Or in this case did not work. It's the business of the college to overrule the voters when in their judgment the voter's judgment needs overruling. That they did not, and at the time there were news reports that suggested that some college members wanted to, suggests a significant failure of the system. It takes courage to stand against the crowd; they didn't have it, even when the popular vote was for Clinton.


(EurActiv, 21 Mar 2019) The Arctic region's co-operation in the battle against global warming by reducing black carbon emissions is being hampered by the United States and Russia, the Finnish foreign ministry said on Wednesday (20 March).
Black carbon, the soot produced by burning fossil fuels and biomass, is an atmospheric pollutant that is particularly problematic in the Arctic region, where it darkens snow and ice, making them retain more warmth and melt faster.
"The United States has pulled out from joint reduction targets and Russia has not submitted its national calculations on black carbon emissions," the Finnish foreign ministry said ahead of the Arctic Council's meeting in Finland over 6-7 May, which US Secretary of State Mike Pompeo is expected to attend.
Finland holds the rotating chairmanship of the Arctic Council, which also includes Canada, Denmark, Iceland, Norway, Russia, Sweden and the United States.
The council coordinates Arctic policy and is gaining clout as sea ice thaws and opens up new trade routes, intensifying competition for its oil, gas and mineral resources while also putting its fragile ecosystem at risk.
"About one third of warming in the Arctic is caused by black carbon emissions of the Arctic Council members," the Finnish foreign ministry's statement said, adding that the majority of its member states had lowered their black carbon emissions.
Two years ago at the council's previous ministerial meeting in Alaska, the member countries agreed to cut their black carbon emissions to 25-33% below 2013 levels by 2025.


Q-Assessor now supports full internationalization of a study in any of — at present — 22 other languages besides English. With a single configuration, investigators can match all of Q-Assessor's own texts to the language they use in the content of the study.
We will continue to add more languages as demand for them arises. We also welcome any suggested edits to our translated texts from native speakers who find issues with our initial translations — which were generated by Google's translation service.
Hi! I'm wondering whether the internationalization supports the Russian language?
… just saw your list; looks like Russian is supported.


Dave was involved in a great hack last week in Perth with Microsoft. The goal was to enable the conversational interface that we have built there via voice, based on Microsoft Cognitive Services.
We used speaker recognition service to enrol a voice, link it to an AD account, do some smarts with tokens to pass authentication credentials around and then used custom speech to load an acoustic model, a custom dictionary and custom pronunciation.
Amazing what you can achieve in a focussed few days.


All families in this study are housed. This study sets out to define and measure inadequate housing, hidden homelessness, and the risk of absolute homelessness in a low-income, housed population. This includes families on a continuum of housing vulnerability and homelessness, from inadequate and precarious housing, to hidden homelessness, to visible homelessness and shelter use, to re-housing after a period in a shelter. Families often move back and forth along this continuum.


If you are looking for an amazingly flavorful yet simple recipe for your next luau, look no further. These taste as great as they look!
In a medium bowl, whisk together the pineapple juice, Hasty-Bake BBQ Sauce, soy sauce, honey, and chili garlic sauce. Reserve 1/4 of a cup for basting.
Add the chicken pieces to the bowl and allow to marinade while you prepare the pineapple, onion, pepper, and jalapeno.
Slice the jalapenos into 1/2 inch slices.
Skewer onto metal skewers in this order: pineapple, jalapeno, red bell pepper, red onion, chicken, pineapple, jalapeno, red bell pepper, red onion, chicken, pineapple.
Grill for about 2-4 minutes, turn 1/4 baste with reserved marinade and grill for about 2-4 minutes, turn 1/4 baste and grill for about 2-4 minutes, turn 1/4 baste and grill for 2-4 minutes until all sides of the chicken are dark golden brown and the fruit/veggies have a nice char.
Serve with grilled pineapple rice.


Other critics used this isolated event, which even provoked considerable local criticism, to issue blanket condemnations of whites, Christianity, Republicans, and, of course, "€œthe South,"€ although the South has always been America's blackest region.
No matter the tribe, that's how all tribes act"€"they seek to dehumanize and eradicate non-members. Why, it's almost like they act that way without even thinking.
No cultural group on Earth seems capable of existing without demonizing and targeting a cultural "€œother."€ Whether it's manifested through blood (race and ethnicity) or water (religion and ideology), tribal behavior seems indelibly identical. Groups can"€™t seem to exist, much less thrive, without a designated scapegoad scapegoat.
Despite all the efforts to "€œget past"€ racism and "€œerace"€ it, all human social groups seem incapable of getting past the same sort of ingroup/outgroup, good guys/bad guys, devils/angels binary thinking for which they condemn those pesky, incestuous, snaggletoothed "€œracists"€"€"who, as luck would have it, always seem to belong to the same race, even though, well, um, race doesn"€™t exist.
If you want to get conspiratorial"€"or at least acknowledge the fact that "divide and conquer" is the oldest trick in the political playbook"€"stuffing a nation full of people who are bound to have tribal differences is a good way to keep them at each other's throats rather than focused on the power mechanisms that control them.
Modern social scientists, who are little more than unscientific socialists, variously view racism as a cancer, a sin, an aberration, and a mental illness rather than a natural and ubiquitous phenomenon. Ignoring the fact that every culture throughout human history has been ethnocentric, they frame racism as an exclusive product of white supremacy, European colonialism, and predatory palefaced capitalism. They also shellac it with a thick gloppy glaze of moralistic condemnation, depicting it as the worst and most destructive of all possible human instincts. The psychological establishment is well on its way to depicting "€œsociobiological"€ drives as personality disorders rather than natural drives toward self-preservation, which in a bygone era were seen as the epitome of mental health.


❶King, Jeannette, and Pam Morris.
The color is repellant, almost revolt ing ; a smouldering unclean yellow, strangely faded by the slow-turning sun light. It is a dull yet lurid orange in some.
The Yellow Wallpaper [Charlotte Perkins Gilman] on directlenders.ml *FREE* shipping on qualifying offers. Book by Gilman, Charlotte Perkins/5().
A summary of Themes in Charlotte Perkins Gilman's The Yellow Wallpaper. Learn exactly what happened in this chapter, scene, or section of The Yellow Wallpaper and what it means. Perfect for acing essays, tests, and . Use our free chapter-by-chapter summary and analysis of The Yellow Wallpaper. It helps middle and high school students understand Charlotte Perkins .
"The Yellow Wallpaper" is an exaggerated account of Charlotte Perkins Gilman's personal experiences. In , shortly after the birth of her daughter, Gilman began to suffer from serious depression and fatigue. She was referred to Silas Weir Mitchell, a leading specialist in women's nervous. Complete summary of Charlotte Perkins Gilman's The Yellow Wallpaper. eNotes plot summaries cover all the significant action of The Yellow Wallpaper.


Regular Monthly Donations that finance recurring expenses at the Centre.
One-Time Donations to raise funds for specific projects or administrative expenses.• Finance from Active Members either by out of pocket expenses, or in kind services.
2. Specific Project Funding from International resources which normally finance Peace-Building projects in conflict areas of the world.


The following is an email response from a friend, whom I asked for some particulars about flying a P-40, needed for a script I was editing. Sadly, I had never flown a P-40, but Jim Appleby certainly had, along with maybe 101 other types in his pile of logbooks.
You can come up with some of the damndest questions! When I was younger, my brain was a lot swifter, but in some 40+ years it doesn't respond very well. So, we'll go from what's left of the memory!
For gear retraction, turn on the electric hydraulic pump located in the baggage compartment area aft, which built up your gear pressure. During WW2 it used a mixture of castor oil and alcohol. Once pressure is built, reach to the left, pull the yellow gear-retraction handle out of the detent and raise to full-up limit. After this you lightly touch the brakes to stop wheel rotation as the snubbers in the wheel well generally don't work too well, and tires eat the hell out of the canvas covers located in the top.
The pump is very inadequate for the job! So to get the gear up, you usually assist with the emergency hand pump located on the right-hand side of the pilot seat, bolted to the top of the wing. Some 40s had gear indicators on the panel, others had two quarter-inch dowels that protruded through the top skin of the wing and slowly disappeared into the wing as the gear came up. If you're in a late model with a retractable tail wheel, you have no way of knowing whether it's up or down without an indicator. The only sure way of knowing the position of the tail wheel is on landing—if you hear an unusual grinding sound and are trailing smoke and bits of tail wheel doors, etc, you can then assume it's not in the correct position.
Once the gear is up, which can take a few days, you return the gear handle to the neutral detent position, which holds the gear up hydraulically, because there are no up-locks. You can double-check the gear-down position by pushing the gear handle beyond the down position and checking the down-lock position. You also have a gear warning horn, if you can hear it over a controller screaming that your gear is not down! This system varies from the early models to the later ones, but is always very slow on retraction. Not only does the strut fold up, but it swivels 90° to align the wheel flush to the chord of the wing, flat!
Fuel is by way of a manual transfer detent located on the fuel selector handle placard. It is not pressure feed nor does it have an electrical pump—the engine-driven pump or the booster pump physically picks up gas out of the tank and feeds it to the engine. You can salvo the belly tank—I don't remember whether this was manual or electrical, but I do recall in post-war civil models, the FAA enforced safety-wiring the drop release where it physically could not be salvoed from the aircraft in flight.
In stretching fuel mileage, the theory is 1600 rpm, 36" manifold pressure, and manually leaning the mixture to give you about 170 mph in cruise (in days before the knot was invented). This is sort of a contradiction. You save a hell of a lot of fuel, but you really don't go anywhere. Whereas with normal cruise power you could be buzzing along at 200, more or less. Of course, if you have a 100-mph tailwind, the first method would be quite acceptable.
Gun energizer switches are hung on a lower sub-panel just above your shins, with your gun sight control, etc. Once activated, the guns are fired from the pistol grip on the stick. (I don't remember being able to select the number of guns activated or the means of doing so.) Incidentally, of interest, your mike radio button is located in the center of the throttle ball, and the cowl flaps have a manual control that looks like the parking brake from a 1928 Packard. Wing flaps are hydraulic and very effective.
A P-40 light on fuel, like all good fighters, should always be three-pointed. I've seen 50-55 mph over the threshold with full flaps to be fairly common and very stable. It's really a friendly bird.
One thing about flying the E, which was shorter, I believe 18" was that in a terminal velocity dive—the plane was redlined at 507 mph! I never had the balls to try it because I always ran out of rudder at about 400, then had to use aileron to keep things going straight. By then you were thoroughly convinced that the next maneuver would be a series of slow rolls to the left. Discretion being the better part of valor, you simply pulled the throttle and stick back slowly and reduced your airspeed.
I would suggest after all this baloney you seek out or locate via the Internet the AN-P40-1 Tech Order, the pilot's operating handbook. That'll tell you all the stuff I forgot.


text:
var http = require('http');
http.createServer(function(request, response){
  response.writeHead(200);
  response.write("Hello, this is dog. ");
  setTimeout(function(){
    response.write("Dog is finished.");
    response.end();
  }, 5000)
}).listen(8080);

redpajama_set_name: RedPajamaGithub


Truman State University has recently partnered with Hoonuit (formerly known as Atomic Learning), award-winning provider of online technology training and support. Through this partnership, Truman is able to provide learning opportunities that make it easy for learners of all ages to embrace technology and develop critical skills for success at school, at work, and in life.
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Truman is dedicated to bringing technology training to all learners on campus through this partnership. With Hoonuit (pronounced "who knew it"), schools can provide instructors, staff, and students 24/7 access to technology training resources. Featuring a guided, step-by-step path through online workshops on topics from APA and MLA to blogging and online discussions, Hoonuit provides valuable educational resources as well as just-in-time training and support on over 215 applications. Hoonuit's online resources simplify campus technology training and support.
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Founded in 2000 by a group of technology educators, Hoonuit serves over 16 million individuals in more than 45 countries worldwide Online and on-demand, Hoonuit's award-winning resources for technology integration and support, student training, and 21st century professional development create flexible training opportunities that make it easy for learners of all ages to embrace technology. Visit Hoonuit online at hoonuit.truman.edu.


Do u believe in Holy Trinity?
Drums in every single church on this planet. What do u say to that?
Has Christianity ever helped u through a rough time?
Do u think Christianity should have so many groups: so many different churches and other Christian communities?
Pick a book of the Bible u like to read?
Which one of those religions is meer similar to Christianity?
Do u believe every single person living on this world has their own guardian angel?
Would u agree that the ecumenism between different churches could lead to greater unity within the reunited & restored modern-day ecumenical Christian Church?
Do u support the ordaining of women in entire Christian world,including the Catholic Church & the Eastern Orthodox churches as well?
Do u interpret the Bible literally? Word for word?
How Often Do u Go To church?
How do u pronounce "amen" in your dialect/language?
Are Atheists and Agnostics evil and deserve eternal Hell just because they don't believe in/question God? Yes of no. Maybe is not an option.
Have u ever helped someone who didn't know Christ?
Are u a strict Christian?
Are u a Jehovah Witness?
Have u see a movie "God's not Dead"?
When u grow up,do u want to be a preacher?
Do u sing in church?
Have u try to help others while sharing about Christ?
Have u ever been attacked door a demon before?
Do u have vrienden that are atheist, deist,satanist,or etc?
Do u believe that evolution is compatible with Christian beliefs?
What dag do u keep as your Sabbath?
Is there a Christianity club at your school?
de keuze van de fan: Yes, but I didn't join.
Do u think the animals will go to Heaven?
GOD zei that if childern still disobey there parants,they needed to be whip door a rod (belt). Do u get whuppings if u do wrong?
Be honest. According to Ephesians it saids not to foul words like cussing. Do u still cuss?
Do u think using the internet to help u on an assignment is cheating?
Do u call him The Devil, Satan, of Lucifer? Just a random Christian question.
Do u have a friend who's a Satanist?
What is your favourite gospel song that u have song ?
Using Christianity as an excuse to bully gays is no excuse.
Do u believe the Bible was created door God?
Anyone here been to Jenness Park/ Fuge Camps anywhere in America? If so wanna kom bij the club?
Did your trust in God save your life of save u from being mentally ill?
What is the regenboog symbol mean to you?
Which of the following is your favoriete version of the bible?
are u a christian ?
Do u take the Bible literally of do u interpret it?
Have u ever heard of Open Doors?
Do u quote scripture when u are trying to prove a point about something dealing with God?
Do u tell others about your God and his son Jesus Christ?
Do u believe in the "Apostles Creed"?
Which theory of Creation do u support of agree with most?
Trivia: Did u know there are Islamic nations mentioned in the Old Testament?
What sect of Christianity are you?
what do ya think will happen when u die?
de keuze van de fan: i'll go to heaven!
if u died into the light,what would u do?
Do u only listen to Christian/Gospel music?
de keuze van de fan: I listen to other genres, including Christian, but not any that displease God.
Do u wanna kom bij my "Jotta A." spot?
de keuze van de fan: Who is he?
Do u wanna kom bij my JJ Heller spot?
Does anyone else hate atheism of is it just me.
de keuze van de fan: Dislike/Disagree: Yes. Hate: No.
Will u kom bij my Heaven club?
Which book of the Bible is meer interesting?
Which of these is your favoriete hymn?
If u converted, which religion did u convert from?
Do u dress modestly as a Christian?
which version of the bible do u prefer?
What, in your opinion, is the meer important factor on going to Heaven?
How should Christmas be celebrated? Exchanging gifts with vrienden and family of partying.
de keuze van de fan: Christmas is to REMEMBER Christ and what he done for us; door exchanging gifts.


Not sure what you're saying here. They way I understood it from Nick is that the formal de_CH version would most like be more used than the informal one currently available and thus would deserve a proper site.
I asked for a de-CH at Ze long time. He always said we are speaking Swiss German, which is why we should take the de-DE version.
I have just imported the formal one for the admin section in dev, too. But for some reason https://translate.wordpress.org/projects/wp/3.9.x/admin is still only showing a blank page.
Regarding Rosetta page: I think the ZIP should contain the formal Swiss version of the .po files. If someone then installs WordPress from the official Swiss German ZIP he/she will also see the Swiss German blog postings on the admin dashboard widgets.
What is the purpose of gsw and de_CH? Please see http://www.ethnologue.com/country/CH/languages for disambiguation. Is it that de_CH is significantly different from both de_DE and gsw? I'm asking because there is no mention in Ethnologue of a version of Swiss German somewehere between Standard German and Schwyzerdütsch. Might this be just a matter of formal vs. informal variants of gsw?
the formal de_CH version would most like be more used than the informal one currently available and thus would deserve a proper site.
I'm not so sure. Other languages have variants (pt_PT and de_DE, at least), and those do not usually warrant a separate site. Remember that an additional site requires additional maintenance. Granted there is the question of packaging releases, which isn't variant-aware, but maybe @nacin has plans or can recommend a sensible approach to this.
for some reason https://translate.wordpress.org/projects/wp/3.9.x/admin is still only showing a blank page.
Looks like a bug to to me, but there's been some activity on the GP repo lately, maybe @yoavf or @markoheijnen can take a look.
The "treating it as a separate site" came from the wish to do proper releases and the expectation the formal version would actually find itself on more installations than the informal.
Regarding the bug, I'm guessing it has something to do with copying dev to 3.9 project in GlotPress?
Consider the 3.9.x project to be non-operational (we're trying out some new branching code in GlotPress). wp/dev is still frozen as the 3.9 branch until we can get it fixed, which should be soon.
I do indeed have near-term (4.0) plans to fix up all release packaging, including locale variants.
I'm maintaining the de_CH language pack for Magento and think it absolutely makes sense to have a separate language pack for de_CH, because not only the spelling is different (ss instead of ß), but also some of the words are different, e.g. Kanton instead of Bundesland.
When building an e-commerce website based on WordPress there's one more aspect that comes into play, i.e. number formatting. You need to configure the de_CH locale in a Swiss store, because floating numbers like 1.25 are formatted the same way as in the US, whereas in Germany and Austria they are formatted with a comma, i.e. 1,25.
I'm not sure how other countries with more than one official language handle this (Switzerland has 4 official languages), but another reason for having our own Rosetta site is the country specific blog postings which are pulled into the "WordPress News" section of the admin dashboard of Swiss German (de_CH) WordPress installations.
If I install the regular German ZIP package of WordPress I see German "WordPress Nachrichten" in the dashboard with events in Hamburg that are not necessarily relevant for Swiss WordPress users. So either the Swiss WordPress community would have to post their stuff on blog.wpde.org (as de.wordpress.org has no blog postings) or on ch.wordpress.org.
So in conclusion the best thing for Switzerland would actually be the Rosetta site ch.wordpress.org for all 4 official languages where you can download 4 different ZIP archives, one for each language. If that does not make sense, the 2nd best option would be de-ch.wordpress.org and if that does not make sense either, we would probably have to create our own "inofficial" domain where we gather the ZIP archives for all 4 official languages and can provide a Swiss WordPress news blog, too.
This argument makes a lot more sense, thank you for that. We'll implement de_CH, but I'll still need it in the proper request format.
This is not doable right now, the best we can do is offer a de-ch.wordpress.org. Not only can we not do "country" Rosetta sites, nor can we do "meta-language" Rosetta sites (think a new fr, de or pt Rosetta site, that would list all variants of a language). That said, the necessity is clear (even if the priority mightn't be) and I'm all for discussing what it would take to have those available.
For French there is French (France), French (Canada), etc. so it could also be named German (Switzerland). Anyway, very glad we can now start building the Rosetta page. Very much appreciated!