Pre-steady-state Kinetic Analysis of a Family D DNA Polymerase from Thermococcus sp. 9°N Reveals Mechanisms for Archaeal Genomic Replication and Maintenance

Family D DNA polymerases (polDs) have been implicated as the major replicative polymerase in archaea, excluding the Crenarchaeota branch, and bear little sequence homology to other DNA polymerase families. Here we report a detailed kinetic analysis of nucleotide incorporation and exonuclease activity for a Family D DNA polymerase from Thermococcus sp. 9°N. Pre-steady-state single-turnover nucleotide incorporation assays were performed to obtain the kinetic parameters, k_pol and K_d, for correct nucleotide incorporation, incorrect nucleotide incorporation, and ribonucleotide incorporation by exonuclease-deficient polD. Correct nucleotide incorporation kinetics revealed a relatively slow maximal rate of polymerization (k_pol ∼2.5 s⁻¹) and especially tight nucleotide binding (K_d(dNTP) ∼1.7 μm), compared with DNA polymerases from Families A, B, C, X, and Y. Furthermore, pre-steady-state nucleotide incorporation assays revealed that polD prevents the incorporation of incorrect nucleotides and ribonucleotides primarily through reduced nucleotide binding affinity. Pre-steady-state single-turnover assays on wild-type 9°N polD were used to examine 3′-5′ exonuclease hydrolysis activity in the presence of Mg²⁺ and Mn²⁺. Interestingly, substituting Mn²⁺ for Mg²⁺ accelerated hydrolysis rates >40-fold (k_exo ≥110 s⁻¹ versus ≥2.5 s⁻¹). Preference for Mn²⁺ over Mg²⁺ in exonuclease hydrolysis activity is a property unique to the polD family. The kinetic assays performed in this work provide critical insight into the mechanisms that polD employs to accurately and efficiently replicate the archaeal genome. Furthermore, despite the unique properties of polD, this work suggests that a conserved polymerase kinetic pathway is present in all known DNA polymerase families.     

Sulfur assimilation in c(4) plants: intercellular compartmentation of adenosine 5'-triphosphate sulfurylase in crabgrass leaves

The activity of adenosine 5′ triphosphate sulfurylase was determined in crabgrass mesophyll cells, bundle sheath strands, and whole leaf extracts. The enzyme was assayed by following molybdate-dependent pyrophosphate release from ATP, ³⁵SO₄²⁻ incorporation into adenosine 5′ phosphosulfate, and ATP synthesis dependent upon adenosine 5′ phosphosulfate and inorganic pyrophosphate. With all assays, greater than 90% of the activity was found in extracts from bundle sheath strands. The activities in whole leaf extracts were consistently intermediate between the activities of mesophyll and bundle sheath extracts and extract-mixing experiments gave no indication of enzyme activation or inhibition in vitro. Whole leaf activities were several hundred-fold less than concurrent measurements of ribulose 1,5-bisphosphate and phosphoenolpyruvate carboxylase activities, which is interpreted as being consistent with the relative amounts of elemental carbon and sulfur found in higher plants. A hypothesis is presented for the intercellular compartmentation of sulfur assimilation in relationship to NO₃⁻ and CO₂ assimilation in leaves of C₄ plants.     

Inhibition of tonoplast ATPase by 2',3'-dialdehyde derivative of ATP

The 2′,3′-dialdehyde derivative of ATP (dial-ATP) has been shown to be an affinity label for the ATP binding site of the H⁺-ATPase from tonoplast of etiolated mung bean seedlings (Vigna radiata L.). The dial-ATP caused marked inactivation of enzymatic activities of both membrane-bound and soluble ATPase and its associated proton translocation. The inactivation was reversible, but could be stabilized by NaBH₄. The sodium dodecyl sulfatepolyacrylamide gel electrophoresis pattern revealed that the dial-ATP binding site was in the large (A) subunit of ATPase. The inhibition could be substantially protected by its physiological substrate ATP, pyrophosphate, and nucleotides in the decreasing order: ATP > pyrophosphate > ADP = AMP > GTP > CTP = UTP. A Lineweaver-Burk plot showed that the mode of inhibition was competitive with respect to ATP. Loss of ATPase activity followed pseudo-first order kinetics with a K_i of 4.1 millimolar, a minimum inactivation half-time of 20 seconds, and a pseudo-first order rate constant of 0.035 s⁻¹. The double logarithmic plot of apparent rate constant versus dial-ATP concentration gave a slope of 0.927, indicating that inactivation results from reaction of at least one lysine residue at the catalytic site of the large subunit. Labeling studies with [³H]dial-ATP indicate that the incorporation of approximately 1 mole of dial-ATP per mole ATPase is sufficient to completely inhibit the ATPase. A working model of nonequivalent subunits for enzymatic mechanism of vacuolar ATPase is suggested.     

Two novel dATP analogs for DNA photoaffinity labeling

Two new photoreactive dATP analogs, N⁶-[4-azidobenzoyl–(2-aminoethyl)]-2′-deoxyadenosine-5′-triphosphate (AB-dATP) and N⁶-[4-[3-(trifluoromethyl)-diazirin-3-yl]benzoyl-(2-aminoethyl)]-2′-deoxyadenosine-5′-triphosphate (DB-dATP), were synthesized from 2′-deoxyadenosine-5′-monophosphate in a six step procedure. Synthesis starts with aminoethylation of dAMP and continues with rearrangement of N¹-(2-aminoethyl)-2′-deoxyadenosine-5′-monophosphate to N⁶-(2-aminoethyl)-2′-deoxyadenosine-5′-monophosphate (N⁶-dAMP). Next, N⁶-dAMP is converted into the triphosphate form by first protecting the N-6 primary amino group before coupling the pyrophosphate. After pyrophosphorylation, the material is deprotected to yield N⁶-(2-aminoethyl)-2′-deoxyadenosine-5′-triphosphate (N⁶-dATP). The N-6 amino group is subsequently used to attach either a phenylazide or phenyldiazirine and the photoreactive nucleotide is then enzymatically incorporated into DNA. N⁶-dATP and its photoreactive analogs AB-dATP and DB-dATP were successfully incorporated into DNA using the exonuclease-free Klenow fragment of DNA polymerase I in a primer extension reaction. UV irradiation of the primer extension reaction with AB-dATP or DB-dATP showed specific photocrosslinking of DNA polymerase I to DNA.     

Detection of Reaction Intermediates during Human Cystathionine β-Synthase-monitored Turnover and H2S Production

Human cystathionine β-synthase (CBS), a novel heme-containing pyridoxal 5′-phosphate enzyme, catalyzes the condensation of homocysteine and serine or cysteine to produce cystathionine and H₂O or H₂S, respectively. The presence of heme in CBS has limited spectrophotometric characterization of reaction intermediates by masking the absorption of the pyridoxal 5′-phosphate cofactor. In this study, we employed difference stopped-flow spectroscopy to characterize reaction intermediates formed under catalytic turnover conditions. The reactions of l-serine and l-cysteine with CBS resulted in the formation of a common aminoacrylate intermediate (k_obs = 0.96 ± 0.02 and 0.38 ± 0.01 mm⁻¹ s⁻¹, respectively, at 24 °C) with concomitant loss of H₂O and H₂S and without detectable accumulation of the external aldimine or other intermediates. Homocysteine reacted with the aminoacrylate intermediate with k_obs = 40.6 ± 3.8 s⁻¹ and re-formed the internal aldimine. In the reverse direction, CBS reacted with cystathionine, forming the aminoacrylate intermediate with k_obs = 0.38 ± 0.01 mm⁻¹ s⁻¹. This study provides the first insights into the pre-steady-state kinetic mechanism of human CBS and indicates that the reaction is likely to be limited by a conformational change leading to product release.     

Ferrocene conjugates of dUTP for enzymatic redox labelling of DNA

Two ferrocene-labelled analogues of dTTP, 5-(3-ferrocenecarboxamidopropenyl-1) 2′-deoxyuridine 5′-triphosphate (Fc1-dUTP) and 5-(3-ferroceneacet-amidopropenyl-1) 2′-deoxyuridine 5′-triphosphate (Fc2-dUTP) have been produced to demonstrate the incorporation of redox labels into DNA by polymerases. Cyclic voltammetry indicates that the ferrocenyl moieties display reversible redox behaviour in aqueous buffer with E_1/2 values of 398 (Fc1-dUTP) and 260 mV (Fc2-dUTP) versus Ag/AgCl. Primer extension by the proofreading enzymes Klenow fragment and T4 DNA polymerase shows that Fc1-dUTP is efficiently incorporated into DNA during synthesis, including incorporation of two successive modified nucleotides. Production of a 998 bp amplicon by Tth DNA polymerase demonstrates that Fc1-dUTP is also a satisfactory substrate for PCR. Despite its structural similarity, Fc2-dUTP acts predominantly as a terminator with the polymerases employed here. UV melting analysis of a 37mer duplex containing five Fc1-dU residues reveals that the labelled nucleotide introduces only a modest helix destabilisation, with T_m = 71 versus 75°C for the corresponding natural construct. Modified DNA is detected at femtomole levels using a HPLC system with a coulometric detector. The availability of simple and effective enzymatic labelling strategies should promote the further development of electrochemical detection in nucleic acid analysis.     

Kinetic mechanism and fidelity of nick sealing by Escherichia coli NAD+-dependent DNA ligase (LigA)

Escherichia coli DNA ligase (EcoLigA) repairs 3′-OH/5′-PO₄ nicks in duplex DNA via reaction of LigA with NAD⁺ to form a covalent LigA-(lysyl-Nζ)–AMP intermediate (step 1); transfer of AMP to the nick 5′-PO₄ to form an AppDNA intermediate (step 2); and attack of the nick 3′-OH on AppDNA to form a 3′-5′ phosphodiester (step 3). A distinctive feature of EcoLigA is its stimulation by ammonium ion. Here we used rapid mix-quench methods to analyze the kinetic mechanism of single-turnover nick sealing by EcoLigA–AMP. For substrates with correctly base-paired 3′-OH/5′-PO₄ nicks, k_step2 was fast (6.8–27 s⁻¹) and similar to k_step3 (8.3–42 s⁻¹). Absent ammonium, k_step2 and k_step3 were 48-fold and 16-fold slower, respectively. EcoLigA was exquisitely sensitive to 3′-OH base mispairs and 3′ N:abasic lesions, which elicited 1000- to >20000-fold decrements in k_step2. The exception was the non-canonical 3′ A:oxoG configuration, which EcoLigA accepted as correctly paired for rapid sealing. These results underscore: (i) how EcoLigA requires proper positioning of the nick 3′ nucleoside for catalysis of 5′ adenylylation; and (ii) EcoLigA''s potential to embed mutations during the repair of oxidative damage. EcoLigA was relatively tolerant of 5′-phosphate base mispairs and 5′ N:abasic lesions.     

Essential Role of the CBD1-CBD2 Linker in Slow Dissociation of Ca2+ from the Regulatory Two-domain Tandem of NCX1

In NCX proteins CBD1 and CBD2 domains are connected through a short linker (3 or 4 amino acids) forming a regulatory tandem (CBD12). Only three of the six CBD12 Ca²⁺-binding sites contribute to NCX regulation. Two of them are located on CBD1 (K_d = ∼0.2 μm), and one is on CBD2 (K_d = ∼5 μm). Here we analyze how the intrinsic properties of individual regulatory sites are affected by linker-dependent interactions in CBD12 (AD splice variant). The three sites of CBD12 and CBD1 + CBD2 have comparable K_d values but differ dramatically in their Ca²⁺ dissociation kinetics. CBD12 exhibits multiphasic kinetics for the dissociation of three Ca²⁺ ions (k_r = 280 s⁻¹, k_f = 7 s⁻¹, and k_s = 0.4 s⁻¹), whereas the dissociation of two Ca²⁺ ions from CBD1 (k_f = 16 s⁻¹) and one Ca²⁺ ion from CBD2 (k_r = 125 s⁻¹) is monophasic. Insertion of seven alanines into the linker (CBD12–7Ala) abolishes slow dissociation of Ca²⁺, whereas the kinetic and equilibrium properties of three Ca²⁺ sites of CBD12–7Ala and CBD1 + CBD2 are similar. Therefore, the linker-dependent interactions in CBD12 decelerate the Ca²⁺ on/off kinetics at a specific CBD1 site by 50–80-fold, thereby representing Ca²⁺ “occlusion” at CBD12. Notably, the kinetic and equilibrium properties of the remaining two sites of CBD12 are “linker-independent,” so their intrinsic properties are preserved in CBD12. In conclusion, the dynamic properties of three sites are specifically modified, conserved, diversified, and integrated by the linker in CBD12, thereby generating a wide range dynamic sensor.     

Kinesin-2 KIF3AB Exhibits Novel ATPase Characteristics

KIF3AB is an N-terminal processive kinesin-2 family member best known for its role in intraflagellar transport. There has been significant interest in KIF3AB in defining the key principles that underlie the processivity of KIF3AB in comparison with homodimeric processive kinesins. To define the ATPase mechanism and coordination of KIF3A and KIF3B stepping, a presteady-state kinetic analysis was pursued. For these studies, a truncated murine KIF3AB was generated. The results presented show that microtubule association was fast at 5.7 μm⁻¹ s⁻¹, followed by rate-limiting ADP release at 12.8 s⁻¹. ATP binding at 7.5 μm⁻¹ s⁻¹ was followed by an ATP-promoted isomerization at 84 s⁻¹ to form the intermediate poised for ATP hydrolysis, which then occurred at 33 s⁻¹. ATP hydrolysis was required for dissociation of the microtubule·KIF3AB complex, which was observed at 22 s⁻¹. The dissociation step showed an apparent affinity for ATP that was very weak (K_½,ATP at 133 μm). Moreover, the linear fit of the initial ATP concentration dependence of the dissociation kinetics revealed an apparent second-order rate constant at 0.09 μm⁻¹ s⁻¹, which is inconsistent with fast ATP binding at 7.5 μm⁻¹ s⁻¹ and a K_d,ATP at 6.1 μm. These results suggest that ATP binding per se cannot account for the apparent weak K_½,ATP at 133 μm. The steady-state ATPase K_m,ATP, as well as the dissociation kinetics, reveal an unusual property of KIF3AB that is not yet well understood and also suggests that the mechanochemistry of KIF3AB is tuned somewhat differently from homodimeric processive kinesins.     

The effect of the 2-amino group of 7,8-dihydro-8-oxo-2'-deoxyguanosine on translesion synthesis and duplex stability

Replication of DNA containing 7,8-dihydro-8-oxo-2′-deoxyguanosine (OxodG) gives rise to G → T transversions. The syn-isomer of the lesion directs misincorporation of 2′-deoxyadenosine (dA) opposite it. We investigated the role of the 2-amino substituent on duplex thermal stability and in replication using 7,8-dihydro-8-oxo-2′-deoxyinosine (OxodI). Oligonucleotides containing OxodI at defined sites were chemically synthesized via solid phase synthesis. Translesion incorporation opposite OxodI was compared with 7,8-dihydro-8-oxo-2′-deoxyguanosine (OxodG), 2′-deoxyinosine (dI) and 2′-deoxyguanosine (dG) in otherwise identical templates. The Klenow exo⁻ fragment of Escherichia coli DNA polymerase I incorporated 2′-deoxyadenosine (dA) six times more frequently than 2′-deoxycytidine (dC) opposite OxodI. Preferential translesion incorporation of dA was unique to OxodI. UV-melting experiments revealed that DNA containing OxodI opposite dA is more stable than when the modified nucleotide is opposed by dC. These data suggest that while duplex DNA accommodates the 2-amino group in syn-OxodG, this substituent is thermally destabilizing and does not provide a kinetic inducement for replication by Klenow exo⁻.     

Two Carotenoid Oxygenases Contribute to Mammalian Provitamin A Metabolism

Mammalian genomes encode two provitamin A-converting enzymes as follows: the β-carotene-15,15′-oxygenase (BCO1) and the β-carotene-9′,10′-oxygenase (BCO2). Symmetric cleavage by BCO1 yields retinoids (β-15′-apocarotenoids, C₂₀), whereas eccentric cleavage by BCO2 produces long-chain (>C₂₀) apocarotenoids. Here, we used genetic and biochemical approaches to clarify the contribution of these enzymes to provitamin A metabolism. We subjected wild type, Bco1^−/−, Bco2^−/−, and Bco1^−/−Bco2^−/− double knock-out mice to a controlled diet providing β-carotene as the sole source for apocarotenoid production. This study revealed that BCO1 is critical for retinoid homeostasis. Genetic disruption of BCO1 resulted in β-carotene accumulation and vitamin A deficiency accompanied by a BCO2-dependent production of minor amounts of β-apo-10′-carotenol (APO10ol). We found that APO10ol can be esterified and transported by the same proteins as vitamin A but with a lower affinity and slower reaction kinetics. In wild type mice, APO10ol was converted to retinoids by BCO1. We also show that a stepwise cleavage by BCO2 and BCO1 with APO10ol as an intermediate could provide a mechanism to tailor asymmetric carotenoids such as β-cryptoxanthin for vitamin A production. In conclusion, our study provides evidence that mammals employ both carotenoid oxygenases to synthesize retinoids from provitamin A carotenoids.     

Adenosine 5'-sulphatophosphate kinase activity in spinach leaf tissue

1. An F⁻-insensitive 3′-nucleotidase was purified from spinach leaf tissue; the enzyme hydrolysed 3′-AMP, 3′-CMP and adenosine 3′-phosphate 5′-sulphatophosphate but not adenosine 5′-nucleotides nor PP_i. The pH optimum of the enzyme was 7.5; K_m (3′-AMP) was approx. 0.8mm and K_m (3′-CMP) was approx. 3.3mm. 3′-Nucleotidase activity was not associated with chloroplasts. Purified Mg²⁺-dependent pyrophosphatase, free from F⁻-insensitive 3′-nucleotidase, catalysed some hydrolysis of 3′-AMP; this activity was F⁻-sensitive. 2. Adenosine 5′-sulphatophosphate kinase activity was demonstrated in crude spinach extracts supplied with 3′-AMP by the synthesis of the sulphate ester of 2-naphthol in the presence of purified phenol sulphotransferase; purified ATP sulphurylase and pyrophosphatase were also added to synthesize adenosine 5′-sulphatophosphate. Adenosine 5′-sulphatophosphate kinase activity was associated with chloroplasts and was released by sonication. 3. Isolated chloroplasts synthesized adenosine 3′-phosphate 5′-sulphatophosphate from sulphate and ATP in the presence of a 3′-nucleotide; the formation of adenosine 5′-sulphatophosphate was negligible. In the absence of a 3′-nucleotide the synthesis of adenosine 3′-phosphate 5′-sulphatophosphate was negligible, but the formation of adenosine 5′-sulphatophosphate was readily detected. Some properties of the synthesis of adenosine 3′-phosphate 5′-sulphatophosphate by isolated chloroplasts are described. 4. Adenosine 3′-phosphate 5′-sulphatophosphate, synthesized by isolated chloroplasts, was characterized by specific enzyme methods, electrophoresis and i.r. spectrophotometry. 5. Isolated chloroplasts catalysed the incorporation of sulphur from sulphate into cystine/cysteine; the incorporation was enhanced by 3′-AMP and l-serine. It was concluded that adenosine 3′-phosphate 5′-sulphatophosphate is an intermediate in the incorporation of sulphur from sulphate into cystine/cysteine.     

Turnip Yellow Mosaic Virus RNA as a Substrate of the Transfer RNA Nucleotidyltransferase II. Incorporation of Cytidine 5'-Monophosphate and Determination of a Short Nucleotide Sequence at the 3' End of the RNA

Turnip yellow mosaic virus (TYMV) RNA treated with snake venom phosphodiesterase accepts cytidine 5′-monophosphate and adenosine 5′-monophosphate (AMP) when it is incubated in the presence of cytidine 5′-triphosphate (CTP), adenosine 5′-triphosphate, and Escherichia coli transfer RNA nucleotidyltransferase; untreated TYMV RNA accepts only AMP. When α ³²PCTP was used for terminal labeling, the nearest neighbor analyses and the anallyses after action of various nucleases showed that the sequence of five nucleotides at the 3′ end of TYMV RNA is: pGpCpApCpC. A nuclease present in commerical preparations of snake venom phosphodiesterase leads to the fragmentation of TYMV RNA, the 3′ end of which is found in a fragment having a sedimentation constant close to 5s.     

Kinetics of Denitrifying Growth by Fast-Growing Cowpea Rhizobia

Two fast-growing strains of cowpea rhizobia (A26 and A28) were found to grow anaerobically at the expense of NO₃⁻, NO₂⁻, and N₂O as terminal electron acceptors. The two major differences between aerobic and denitrifying growth were lower yield coefficients (Y) and higher saturation constants (K_s) with nitrogenous oxides as electron acceptors. When grown aerobically, A26 and A28 adhered to Monod kinetics, respectively, as follows: K_s, 3.4 and 3.8 μM; Y, 16.0 and 14.0 g · cells eq⁻¹; μ_max, 0.41 and 0.33 h⁻¹. Yield coefficients for denitrifying growth ranged from 40 to 70% of those for aerobic growth. Only A26 adhered to Monod kinetics with respect to growth on all three nitrogenous oxides. The apparent K_s values were 41, 270, and 460 μM for nitrous oxide, nitrate, and nitrite, respectively; the K_s for A28 grown on nitrate was 250 μM. The results are kinetically and thermodynamically consistent in explaining why O₂ is the preferred electron acceptor. Although no definitive conclusions could be drawn regarding preferential utilization of nitrogenous oxides, nitrite was inhibitory to both strains and effected slower growth. However, growth rates were identical (μ_max, 0.41 h⁻¹) when A26 was grown with either O₂ or NO₃⁻ as an electron acceptor and were only slightly reduced when A28 was grown with NO₃⁻ (0.25 h⁻¹) as opposed to O₂ (0.33 h⁻¹).     

TDP1 repairs nuclear and mitochondrial DNA damage induced by chain-terminating anticancer and antiviral nucleoside analogs

Chain-terminating nucleoside analogs (CTNAs) that cause stalling or premature termination of DNA replication forks are widely used as anticancer and antiviral drugs. However, it is not well understood how cells repair the DNA damage induced by these drugs. Here, we reveal the importance of tyrosyl–DNA phosphodiesterase 1 (TDP1) in the repair of nuclear and mitochondrial DNA damage induced by CTNAs. On investigating the effects of four CTNAs—acyclovir (ACV), cytarabine (Ara-C), zidovudine (AZT) and zalcitabine (ddC)—we show that TDP1 is capable of removing the covalently linked corresponding CTNAs from DNA 3′-ends. We also show that Tdp1^−/− cells are hypersensitive and accumulate more DNA damage when treated with ACV and Ara-C, implicating TDP1 in repairing CTNA-induced DNA damage. As AZT and ddC are known to cause mitochondrial dysfunction, we examined whether TDP1 repairs the mitochondrial DNA damage they induced. We find that AZT and ddC treatment leads to greater depletion of mitochondrial DNA in Tdp1^−/− cells. Thus, TDP1 seems to be critical for repairing nuclear and mitochondrial DNA damage caused by CTNAs.     

Effects of Abiotic Factors on Acetylene Reduction by Cyanobacteria Epiphytic on Moss at a Subantarctic Island

Acetylene reduction (AR) rates by cyanobacteria epiphytic on a moss at Marion Island (46°54′ S, 37°45′ E) increased from −5°C to a maximum at 25 to 27°C. Q₁₀ values between 0 and 25°C were between 2.3 and 2.9, depending on photosynthetic photon flux density. AR rates declined sharply at temperatures above the optimum and were lower at 35°C than at 0°C. Photosynthetic photon flux density at low levels markedly influenced AR, and half of the maximum rate occurred at 84 μmol m⁻² s⁻¹, saturation occurring at ca. 1,000 μmol m⁻² s⁻¹. Higher photosynthetic photon flux density levels decreased AR rates. AR increased up to the highest sample moisture content investigated (3,405%), and the pH optimum was between 5.9 and 6.2. The addition of P, Co, and Mo, individually or together, depressed AR.     

Light-Modulated Responses of Growth and Photosynthetic Performance to Ocean Acidification in the Model Diatom Phaeodactylum tricornutum

Ocean acidification (OA) due to atmospheric CO₂ rise is expected to influence marine primary productivity. In order to investigate the interactive effects of OA and light changes on diatoms, we grew Phaeodactylum tricornutum, under ambient (390 ppmv; LC) and elevated CO₂ (1000 ppmv; HC) conditions for 80 generations, and measured its physiological performance under different light levels (60 µmol m⁻² s⁻¹, LL; 200 µmol m⁻² s⁻¹, ML; 460 µmol m⁻² s⁻¹, HL) for another 25 generations. The specific growth rate of the HC-grown cells was higher (about 12–18%) than that of the LC-grown ones, with the highest under the ML level. With increasing light levels, the effective photochemical yield of PSII (F_v′/F_m′) decreased, but was enhanced by the elevated CO₂, especially under the HL level. The cells acclimated to the HC condition showed a higher recovery rate of their photochemical yield of PSII compared to the LC-grown cells. For the HC-grown cells, dissolved inorganic carbon or CO₂ levels for half saturation of photosynthesis (K_1/2 DIC or K_1/2 CO₂) increased by 11, 55 and 32%, under the LL, ML and HL levels, reflecting a light dependent down-regulation of carbon concentrating mechanisms (CCMs). The linkage between higher level of the CCMs down-regulation and higher growth rate at ML under OA supports the theory that the saved energy from CCMs down-regulation adds on to enhance the growth of the diatom.     

Identification of a 5′-Deoxyadenosine Deaminase in Methanocaldococcus jannaschii and Its Possible Role in Recycling the Radical S-Adenosylmethionine Enzyme Reaction Product 5′-Deoxyadenosine

We characterize here the MJ1541 gene product from Methanocaldococcus jannaschii, an enzyme that was annotated as a 5′-methylthioadenosine/S-adenosylhomocysteine deaminase (EC 3.5.4.31/3.5.4.28). The MJ1541 gene product catalyzes the conversion of 5′-deoxyadenosine to 5′-deoxyinosine as its major product but will also deaminate 5′-methylthioadenosine, S-adenosylhomocysteine, and adenosine to a small extent. On the basis of these findings, we are naming this new enzyme 5′-deoxyadenosine deaminase (DadD). The K_m for 5′-deoxyadenosine was found to be 14.0 ± 1.2 μM with a k_cat/K_m of 9.1 × 10⁹ M⁻¹ s⁻¹. Radical S-adenosylmethionine (SAM) enzymes account for nearly 2% of the M. jannaschii genome, where the major SAM derived products is 5′-deoxyadenosine. Since 5′-dA has been demonstrated to be an inhibitor of radical SAM enzymes; a pathway for removing this product must be present. We propose here that DadD is involved in the recycling of 5′-deoxyadenosine, whereupon the 5′-deoxyribose moiety of 5′-deoxyinosine is further metabolized to deoxyhexoses used for the biosynthesis of aromatic amino acids in methanogens.     

Human DNA Polymerase ν Catalyzes Correct and Incorrect DNA Synthesis with High Catalytic Efficiency

DNA polymerase ν (pol ν) is a low fidelity A-family polymerase with a putative role in interstrand cross-link repair and homologous recombination. We carried out pre-steady-state kinetic analysis to elucidate the kinetic mechanism of this enzyme. We found that the mechanism consists of seven steps, similar that of other A-family polymerases. pol ν binds to DNA with a K_d for DNA of 9.2 nm, with an off-rate constant of 0.013 s⁻¹and an on-rate constant of 14 μm⁻¹ s⁻¹. dNTP binding is rapid with K_d values of 20 and 476 μm for the correct and incorrect dNTP, respectively. Pyrophosphorylation occurs with a K_d value for PP_i of 3.7 mm and a maximal rate constant of 11 s⁻¹. Pre-steady-state kinetics, examination of the elemental effect using dNTPαS, and pulse-chase experiments indicate that a rapid phosphodiester bond formation step is flanked by slow conformational changes for both correct and incorrect base pair formation. These experiments in combination with computer simulations indicate that the first conformational change occurs with rate constants of 75 and 20 s⁻¹; rapid phosphodiester bond formation occurs with a K_eq of 2.2 and 1.7, and the second conformational change occurs with rate constants of 2.1 and 0.5 s⁻¹, for correct and incorrect base pair formation, respectively. The presence of a mispair does not induce the polymerase to adopt a low catalytic conformation. pol ν catalyzes both correct and mispair formation with high catalytic efficiency.