Direct estimation of base-pair exchange kinetics in oligo-DNA by a combination of NOESY and ROESY experiments.

A new method for the determination of the kinetics of exchange of the imino protons of DNA duplex is reported using a combination NOESY and ROESY experiments at short mixing times (< or = 20 ms). These results have been compared with the commonly used longitudinal relaxation approach through the T1 measurement. To calculate kex and pi ex by ROESY-NOESY experiment, the volume of the cross-peaks between imino protons and water in the NOESY and ROESY spectra have been measured separately from the magnetization term. This work shows that the present approach for the measurement of the kinetics of slow exchanging imino protons of DNA duplex is comparable to the saturation recovery experiment in which the exchange rate can be accelerated by the addition of a base catalyst. The present ROESY-NOESY approach has been found to be particularly useful and reasonably accurate for the measurement of exchange kinetics of both the fast- and slow-exchanging imino protons in DNA duplex both under non-physiological and physiological condition where the saturation recovery method can not be used.     

Crystal structure of Nsp15 endoribonuclease NendoU from SARS‐CoV‐2

Severe Acute Respiratory Syndrome coronavirus 2 (SARS‐CoV‐2) is rapidly spreading around the world. There is no existing vaccine or proven drug to prevent infections and stop virus proliferation. Although this virus is similar to human and animal SARS‐CoVs and Middle East Respiratory Syndrome coronavirus (MERS‐CoVs), the detailed information about SARS‐CoV‐2 proteins structures and functions is urgently needed to rapidly develop effective vaccines, antibodies, and antivirals. We applied high‐throughput protein production and structure determination pipeline at the Center for Structural Genomics of Infectious Diseases to produce SARS‐CoV‐2 proteins and structures. Here we report two high‐resolution crystal structures of endoribonuclease Nsp15/NendoU. We compare these structures with previously reported homologs from SARS and MERS coronaviruses.     

Fatty acids of microalgae: diversity and applications

Reviews in Environmental Science and Bio/Technology - The possibility of obtaining commercially valuable products from microalgae stimulates scientific research in this direction. The ability of...     

A Single Carbocyclic Nucleotide Substitution in a 12mer DNA Gives a Hoogsteen Basepaired Duplex (Till 38°C) and a Hairpin (Till 65°C). A 600 MHz NMR Spectroscopic Study

Abstract

The impact of intramolecular stereoelectronic effects has been examined by comparison of the solution structures of natural oligo-DNA duplex, 5′(¹C²G³C⁴G⁵A⁶A⁷T⁸T⁹C¹⁰G¹¹C¹²G)₂ ³′, and its carbocyclic-nucleotide analogues in which the pentose sugar in 2′-dA residue is replaced with its carbocyclic counterpart (i.e. 2′-deoxyaristeromycin). Based on the NMR evidences, it has been shown, that 2′-deoxyaristeromycin analog exists in a dynamic equilibrium between the two forms of duplexes, one with W-C bp and the second with Hoogsteen bp in ca 1:1 ratio at lower temperature (below 35°C) and as hairpin at higher temperature (from ～40° – 60°C).     

Elucidation of the Origin of NOEs And ROEs that Show the Hydration in the Minor and Major Grooves of DNA Duplex with ATTAAT Tract by a Combination of NOESY and ROESY Experiments

Abstract

A combination of NOESY and ROESY experiments (using ammonia as a catalyst across the pH range of 5 to 8.6) has given us a clear understanding regarding the origin of nOes that are attributed to the stereochemical location and the residence time of water in the major and the minor grooves of d⁵'(¹C²C³A⁴T⁵T⁶A⁷A⁸T⁹G¹⁰G)₂ ³' duplex Our conclusions are the following: (i) In the major groove, the presence of ammonia in the buffer does not influence on the process of exchange between bound and bulk water, (ii) It has been found that the observation of the bound water in the minor groove is a result of straight dipole-dipole effect at the physiological pH. (iii) The residence time of water near H2 of adenine (H2A) in the minor groove has been estimated to be in the range of 0.3–0.5ns, which is closer to the residence time of the bound water found on the surface of protein, (iv) The hydration pattern in the minor groove in the physiological pH, under our NMR measurement condition, is similar to the ones found in the X-ray structure, (v) It has been shown that at pH > 8.0 the nOe/rOe intensities of the water-H2A crosspeaks dramatically increase due to dipole-dipole and/or relayed magnetization transfer from H2A to water through ammonia catalyst.     

The stereochemistry of benzo[a]pyrene-2'-deoxyguanosine adducts affects DNA methylation by SssI and HhaI DNA methyltransferases

The biologically most significant genotoxic metabolite of the environmental pollutant benzo[a]pyrene (B[a]P), (+)-7R,8S-diol 9S,10R-epoxide, reacts chemically with guanine in DNA, resulting in the predominant formation of (+)-trans-B[a]P-N(2)-dG and, to a lesser extent, (+)-cis-B[a]P-N(2)-dG adducts. Here, we compare the effects of the adduct stereochemistry and conformation on the methylation of cytosine catalyzed by two purified prokaryotic DNA methyltransferases (MTases), SssI and HhaI, with the lesions positioned within or adjacent to their CG and GCGC recognition sites, respectively. The fluorescence properties of the pyrenyl residues of the (+)-cis-B[a]P-N(2)-dG and (+)-trans-B[a]P-N(2)-dG adducts in complexes with MTases are enhanced, but to different extents, indicating that aromatic B[a]P residues are positioned in different microenvironments in the DNA-protein complexes. We have previously shown that the (+)-trans-isomeric adduct inhibits both the binding and methylating efficiencies (k(cat)) of both MTases [Subach OM, Baskunov VB, Darii MV, Maltseva DV, Alexandrov DA, Kirsanova OV, Kolbanovskiy A, Kolbanovskiy M, Johnson F, Bonala R, et al. (2006) Biochemistry45, 6142-6159]. Here we show that the stereoisomeric (+)-cis-B[a]P-N(2)-dG lesion has only a minimal effect on the binding of these MTases and on k(cat). The minor-groove (+)-trans adduct interferes with the formation of the normal DNA minor-groove contacts with the catalytic loop of the MTases. However, the intercalated base-displaced (+)-cis adduct does not interfere with the minor-groove DNA-catalytic loop contacts, allowing near-normal binding of the MTases and undiminished k(cat) values.     

Construction and characterization of two recombinant bacteria that grow on ortho- and para-substituted chlorobiphenyls

Cloning and expression of the aromatic ring dehalogenation genes in biphenyl-growing, polychlorinated biphenyl (PCB)-cometabolizing Comamonas testosteroni VP44 resulted in recombinant pathways allowing growth on ortho- and para-chlorobiphenyls (CBs) as a sole carbon source. The recombinant variants were constructed by transformation of strain VP44 with plasmids carrying specific genes for dehalogenation of chlorobenzoates (CBAs). Plasmid pE43 carries the Pseudomonas aeruginosa 142 ohb genes coding for the terminal oxygenase (ISPOHB) of the ortho-halobenzoate 1,2-dioxygenase, whereas plasmid pPC3 contains the Arthrobacter globiformis KZT1 fcb genes, which catalyze the hydrolytic para-dechlorination of 4-CBA. The parental strain, VP44, grew only on low concentrations of 2- and 4-CB by using the products from the fission of the nonchlorinated ring of the CBs (pentadiene) and accumulated stoichiometric amounts of the corresponding CBAs. The recombinant strains VP44(pPC3) and VP44(pE43) grew on, and completely dechlorinated high concentrations (up to 10 mM), of 4-CBA and 4-CB and 2-CBA and 2-CB, respectively. Cell protein yield corresponded to complete oxidation of both biphenyl rings, thus confirming mineralization of the CBs. Hence, the use of CBA dehalogenase genes appears to be an effective strategy for construction of organisms that will grow on at least some congeners important for remediation of PCBs.     

Pattern of changes in clinico-immunological indicators of patients with drug allergy during treatment with autologous blood

An efficient method for the treatment of drug allergy was proposed and practically implemented. The method consists in autologous venous blood being lyzed with sterile bidistilled water at a ratio of 5:1 and injected subcutaneously and partially intradermally into the reflexogenic zones of the back 2-3 cm from the spinal column. During the first week of treatment, increasing doses were injected (3 to 10 ml), whereas during the second week the doses decreased from 10 to 3 ml. Following treatment the patients felt better and featured enhanced working ability as well as markedly declined susceptibility to common colds. The most informative immunological indicators were chosen to evaluate the efficiency of therapy.     

Interaction of murine Dnmt3a with DNA containing O<Superscript>6</Superscript>-methylguanine

O⁶-Methylguanine (O⁶meG) is one of the most toxic, mutagenic, and carcinogenic lesions caused by the interaction of DNA with several catabolism products as well as with environmental methylating agents. Carcinogenic impact of O⁶meG can be conditioned not only by its mutagenic properties but also by alteration in enzymatic methylation of the C5 carbon atom of cytosine residue in CpG sequences. In this study, the effect of O⁶meG on DNA methylation by the catalytic domain of murine DNA methyltransferase (MTase) Dnmt3a (Dnmt3a-CD) is assessed. Damaged DNA duplexes cooperatively bind with Dnmt3a-CD, and O⁶meG changes the stability of enzyme-substrate complexes. Kinetic analysis of the methylation reaction revealed that O⁶meG varies the ratio of productive and nonproductive enzyme-substrate complexes and, depending on localization in substrate, causes decrease or increase in DNA methylation. Dnmt3a-CD is less sensitive to the presence of O⁶meG in DNA substrate than procaryotic MTase SssI recognizing CpG.