Recognition of template-primer and gapped DNA substrates by the human DNA polymerase beta

Interactions between human DNA polymerase beta and the template-primer, as well as gapped DNA substrates, have been studied using quantitative fluorescence titration and analytical ultracentrifugation techniques. In solution, human pol beta binds template-primer DNA substrates with a stoichiometry much higher than predicted on the basis of the crystallographic structure of the polymerase-DNA complex. The obtained stoichiometries can be understood in the context of the polymerase affinity for the dsDNA and the two ssDNA binding modes, the (pol beta)(16) and (pol beta)(5) binding modes, which differ by the number of nucleotide residues occluded by the protein in the complex. The analysis of polymerase binding to different template-primer substrates has been performed using the statistical thermodynamic model which accounts for the existence of different ssDNA binding modes and has allowed us to extract intrinsic spectroscopic and binding parameters. The data reveal that the small 8 kDa domain of the enzyme can engage the dsDNA in interactions, downstream from the primer, in both (pol beta)(16) and (pol beta)(5) binding modes. The affinity, as well as the stoichiometry of human pol beta binding to the gapped DNAs is not affected by the decreasing size of the ssDNA gap, indicating that the enzyme recognizes the ssDNA gaps of different sizes with very similar efficiency. On the basis of the obtained results we propose a plausible model for the gapped DNA recognition by human pol beta. The enzyme binds the ss/dsDNA junction of the gap, using its 31 kDa domain, with slight preference over the dsDNA. Binding only to the junction, but not to the dsDNA, induces an allosteric conformational transition of the enzyme and the entire enzyme-DNA complex which results in binding of the 8 kDa domain with the dsDNA. This, in turn, leads to the significant amplification of the enzyme affinity for the gap over the surrounding dsDNA, independent of the gap size. The presence of the 5'-terminal phosphate, downstream from the primer, has little effect on the affinity, but profoundly affects the ssDNA conformation in the complex. The significance of these results for the mechanistic model of the functioning of human pol beta is discussed.     

HIV Rev self-assembly is linked to a molten-globule to compact structural transition

By regulating the differential expression of proviral pre mRNA in the host cell, Rev plays a crucial role in the HIV-1 life cycle. The capacity of Rev to function is intimately linked to its ability to self-associate. Nevertheless, little is known about the exact role of self-association in the molecular mechanism defining its biological activity. A prerequisite knowledge is a definition of the molecular events undertaken by Rev during the process of self-assembly. Thus, this study was initiated to monitor the structure of Rev as a function of protein concentration. Rev undergoes a structural transition as a consequence of self-assembly. This structural transition was monitored by three spectroscopic methods. The accessibility of the single tryptophan in Rev monomer to acrylamide quenching increases with decreasing protein concentration. At very low concentration of Rev, the tryptophan accessibility is close to that of an unfolded Rev. As evaluated by circular dichroism, the secondary structure of Rev is protein concentration dependent as evidenced by an increase in the magnitude of ellipticity with increasing protein concentration. Further, results from ANS binding studies indicate that the ANS binding sites in Rev experience an apparent increase in hydrophobicity as the Rev concentration was increased. These concentration dependent changes seem to reach a maximum above 5 microM Rev monomer concentration. In order to define the mode of Rev self-association sedimentation velocity and equilibrium experiments were conducted. There are evidently two consecutive progressive association processes. At protein concentrations below 0.5 mg/ml, the data from sedimentation studies can be fitted to a single isodesmic model. Simulation of velocity sedimentation profile indicates that free Rev monomer that has not entered into the association processes can best be described to exhibit a value of S(20,w) that is substantially smaller than 1.4 S, a value needed to fit the rest of the data. The latter value is consistent for a Rev monomer with the expected molecules weight and if it were to assume a compact globular shape. These spectroscopic and hydrodynamic results imply that monomeric Rev is in a molten globule state, which becomes more compact upon self-association.     

Synthesis, X-ray crystal structure, antimicrobial activity and photodynamic effects of some thiabendazole complexes

An interesting series of metal complexes of thiabendazole (tbz) is synthesized and characterized by elemental analyses and spectroscopic studies. The crystal structure of the hydrogen bonded one dimensional Co(II) complex, namely [Co(tbz)(2)(NO(3))(H(2)O)](NO(3)) is solved by single crystal X-ray diffraction. The complex crystallizes in monoclinic space group P2(1)/a with unit cell parameters, a=14.366(2), b=11.459(4), c=15.942(3) A, beta=113.78(3) degrees and z=4. The unit cell packing reveals an extensive hydrogen bonding involving a water molecule, nitrate ligands and the protonated nitrogen atoms of the tbz ligands, resulting in a one dimensional hydrogen bonding pattern. The antimicrobial activity of the complexes against selected bacteria (Escherichia coli and Bacillus subtilis) and yeast (Aspergillus flavues) is estimated. The relationship between the enzymatic production of ROS and antimicrobial activity of the complexes is examined, and a good correlation between two factors is found. Photodynamic quantum yields of singlet oxygen production (RNO bleaching assay) and rate of superoxide generation (SOD inhibitable ferricytochrome c reduction assay and EPR spin trapping experiments using 5,5-dimethyl-1-pyrroline-N-oxide as spin trap) by the metal complexes have been studied.     

EF-G-independent reactivity of a pre-translocation-state ribosome complex with the aminoacyl tRNA substrate puromycin supports an intermediate (hybrid) state of tRNA binding

Following peptide-bond formation, the mRNA:tRNA complex must be translocated within the ribosomal cavity before the next aminoacyl tRNA can be accommodated in the A site. Previous studies suggested that following peptide-bond formation and prior to EF-G recognition, the tRNAs occupy an intermediate (hybrid) state of binding where the acceptor ends of the tRNAs are shifted to their next sites of occupancy (the E and P sites) on the large ribosomal subunit, but where their anticodon ends (and associated mRNA) remain fixed in their prepeptidyl transferase binding states (the P and A sites) on the small subunit. Here we show that pre-translocation-state ribosomes carrying a dipeptidyl-tRNA substrate efficiently react with the minimal A-site substrate puromycin and that following this reaction, the pre-translocation-state bound deacylated tRNA:mRNA complex remains untranslocated. These data establish that pre-translocation-state ribosomes must sample or reside in an intermediate state of tRNA binding independent of the action of EF-G.     

Genetic association analysis of KCNQ3 and juvenile myoclonic epilepsy in a South Indian population

Juvenile myoclonic epilepsy (JME) is a common subtype of idiopathic generalized epilepsy that shows a complex pattern of inheritance. We have tested the association between JME phenotype and an intragenic marker in KCNQ3 by using the transmission disequilibrium test in 119 probands and their parents. Mutations in KCNQ3 are known to cause benign familial neonatal convulsions and are involved in the physiologically important M current in neurons. Our results provide suggestive evidence of allelic association between JME and KCNQ3 (P-value=0.008) and raise an interesting possibility of a genetic contribution to JME, viz., of a gene that causes a monogenic form of human epilepsy.     

Real-time quantitative PCR assay on bacterial DNA: In a model soil system and environmental samples

Real-time quantitative PCR (RTQ-PCR) was used to quantify the bacterial target DNA extracted by three commonly used DNA extraction protocols (bead mill homogenization, grinding in presence of liquid nitrogen and hot detergent SDS based enzymatic lysis). For the purpose of our study, pure culture of Bacillus cereus (model organism), sterilized soil seeded with a known amount of B. cereus (model soil system) and samples from woodland and grassland (environmental samples) were chosen to extract DNA by three different protocols. The extracted DNA was then quantified by RTQ-PCR using 16S rDNA specific universal bacterial primers. The standard curve used for the quantification by RTQ-PCR was linear and revealed a strong linear relationship (r(2)=0.9968) with a higher amplification efficiency, e5=1.02. High resolution gel electrophoresis was also carried out to observe the effect of these extraction methods on diversity analysis. For the model soil system, the liquid nitrogen method showed the highest target DNA copy number (1.3 x 10(9) copies/microl). However, for both the environmental samples, the bead beating method was found to be suitable on the basis of the high target DNA copy numbers (5.38 x 10(9) and 4.01 x 10(8) copies/ml for woodland and grassland respectively), high yield (6.4 microg/g and 1.76 microg/g of soil for woodland and grassland respectively) and different band patterns on high resolution gel electrophoresis suggesting an overall high extraction efficiency. This difference in the extraction efficiency between the model soil system and environmental samples may be attributed to different affinity of seeded and native DNA to soil particles.     

Photodynamic effects of two hydroxyanthraquinones

The aim of this work was to investigate the photodynamic action of electron-rich anthraquinones, viz., cynodontin (CYN) and cynodontin-5,8-dimethylether (CYNM). Both optical and EPR methods are used to detect the generation of singlet oxygen. Based on RNO bleaching, relative to rose bengal (RB), singlet oxygen generating efficiencies of CYN and CYNM are derived to be 0.055 and 0.254, respectively. The formation of superoxide anion via electron transfer to O2 was monitored by optical spectroscopy, using SOD-inhibitable cytochrome c reduction assay. The production of O2-* is enhanced in the presence of electron donors such as EDTA and NADH. Photolysis of CYN and CYNM in DMSO, in the presence of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), generates a multi-line EPR spectrum, characteristic of spin adduct mixture of O2-* and *OH. Both optical and ESR measurements indicate that O2-* (Type I) and 1O2 (Type II) paths are involved in CYN and CYNM photodynamic action.     

The cytotoxic activity of ribosome-inactivating protein saporin-6 is attributed to its rRNA N-glycosidase and internucleosomal DNA fragmentation activities

Saporin-6 produced by the plant Saponaria officinalis belongs to the family of single chain ribosome-inactivating proteins. It potently inhibits protein synthesis in eukaryotic cells, by cleaving the N-glycosidic bond of a specific adenine in 28 S rRNA, which results in the cell death. Saporin-6 has also been shown to be active on DNA and induces apoptosis. In the current study, we have investigated the roles of rRNA depurination and the activity of saporin-6 on genomic DNA in its cytotoxic activity. The role of putative active site residues, Tyr(72), Tyr(120), Glu(176), Arg(179), and Trp(208), and two invariant residues, Tyr(16) and Arg(24), proposed to be important for structural stability of saporin-6, has been investigated in its catalytic and cytotoxic activity. These residues were mutated to alanine to generate seven mutants, Y16A, R24A, Y72A, Y120A, E176A, R179A, and W208A. We show that for the RNA N-glycosidase activity of saporin-6, residues Tyr(16), Tyr(72), and Arg(179) are absolutely critical; Tyr(120) and Glu(176) can be partially dispensed with, whereas Trp(208) and Arg(24) do not appear to be involved in this activity. The residues Tyr(72), Tyr(120), Glu(176), Arg(179), and Trp(208) were found to be essential for the genomic DNA fragmentation activity, whereas residues Tyr(16) and Arg(24) do not appear to be required for the DNA fragmentation. The study shows that saporin-6 possesses two catalytic activities, namely RNA N-glycosidase and genomic DNA fragmentation activity, and for its complete cytotoxic activity both activities are required.     

Extracellular domains,transmembrane segments,and intracellular domains interact to determine the cation selectivity of Na,K- and gastric H,K-ATPase

We have previously reported that three residues of the fourth transmembrane segment (TM4) of the Na,K- and gastric H,K-ATPase alpha-subunits appear to play a major role in the distinct cation selectivities of these pumps [Mense, M., et al. (2000) J. Biol. Chem. 275, 1749-1756]. Substituting these three residues in the Na,K-ATPase sequence with their H,K-ATPase counterparts (L319F, N326Y, T340S) and replacing the TM3-TM4 ectodomain sequence with that of the H,K-ATPase alpha-subunit result in a pump that exhibits 50% of its maximal ATPase activity in the absence of Na(+) when the assay is performed at pH 6.0. This effect is not seen when the ectodomain alone is replaced. To gain more insight into the contributions of the three residues to establishing the selectivity of these pumps for Na(+) ions versus protons, we generated Na,K-ATPase constructs in which these residues are replaced by their H,K-ATPase counterparts either singly or in combinations. Surprisingly, none of the point mutants nor even the triple mutant was able to hydrolyze ATP at pH 6.0 at a rate greater than 20% of their respective V(max)s. For the point mutants L319F and N326Y, protons seem to competitively inhibit ATP hydrolysis at pH 6.0, based on the low apparent affinity for Na(+) ions at pH 6.0 compared to pH 7.5. It would appear, therefore, that the cation selectivity of Na,K- and H,K-ATPase is generated through a cooperative effort between residues of transmembrane segments and the flanking loops that connect these transmembrane domains. This view is further supported by homology modeling of the Na,K-ATPase based on the crystal structure of the SERCA pump.     

Management of root-knot nematode in tomato Lycopersicon esculentum,Mill, with biogas slurry

The effect of biogas slurry application on the severity of root-knot nematode, Meloidogyne incognita, attack on tomato cv. Co-1, was tested in the green house with two levels of biogas slurry: 5% and 10% (w/w) added to soil. Both the number (3 fruits/plant) and fruit yield (35.2 g/plant) of tomato increased significantly with 10% (w/w) biogas slurry. The plants amended with biogas slurry put up more vegetative growth and tended to flower and fruit much earlier than did those of the control. The nematode population in the soil decreased thus decreasing the severity of nematode attack.