Mycobacterium tuberculosis UvrD1 and UvrA Proteins Suppress DNA Strand Exchange Promoted by Cognate and Noncognate RecA Proteins

DNA helicases are present in all kingdoms of life and play crucial roles in processes of DNA metabolism such as replication, repair, recombination, and transcription. To date, however, the role of DNA helicases during homologous recombination in mycobacteria remains unknown. In this study, we show that Mycobacterium tuberculosis UvrD1 more efficiently inhibited the strand exchange promoted by its cognate RecA, compared to noncognate Mycobacterium smegmatis or Escherichia coli RecA proteins. The M. tuberculosis UvrD1(Q276R) mutant lacking the helicase and ATPase activities was able to block strand exchange promoted by mycobacterial RecA proteins but not of E. coil RecA. We observed that M. tuberculosis UvrA by itself has no discernible effect on strand exchange promoted by E. coli RecA but impedes the reaction catalyzed by the mycobacterial RecA proteins. Our data also show that M. tuberculosis UvrA and UvrD1 can act together to inhibit strand exchange promoted by mycobacterial RecA proteins. Taken together, these findings raise the possibility that UvrD1 and UvrA might act together in vivo to counter the deleterious effects of RecA nucleoprotein filaments and/or facilitate the dissolution of recombination intermediates. Finally, we provide direct experimental evidence for a physical interaction between M. tuberculosis UvrD1 and RecA on one hand and RecA and UvrA on the other hand. These observations are consistent with a molecular mechanism, whereby M. tuberculosis UvrA and UvrD1, acting together, block DNA strand exchange promoted by cognate and noncognate RecA proteins.

New evidences on Tau-DNA interactions and relevance to neurodegeneration

Tau is mainly distributed in cytoplasm and also found to be localized in the nucleus. There is limited data on DNA binding potential of Tau.We provide novel evidence on nicking of DNA by Tau. Tau nicks the supercoiled DNA leading to open circular and linear forms. The metal ion magnesium (a co-factor for endonuclease) enhanced the Tau DNA nicking ability, while an endonuclease specific inhibitor,aurinetricarboxylic acid (ATA) inhibited the Tau DNA nicking ability Further, we also evidenced that Tau induces B-C-A mixed conformational transition in DNA and also changes DNA stability. Tau-scDNA complex is more sensitive to DNAse I digestion indicating stability changes in DNA caused by Tau. These findings indicate that Tau alters DNA helicity and integrity and also nicks the DNA. The relevance of these novel intriguing findings regarding the role Tau in neuronal dysfunction is discussed. (C) 2010 Elsevier Ltd. All rights reserved.

Antibacterial & antiplasmid activities of Helicteres isora L.

Background & objectives: The multiple drug resistance (MDR) is a serious health problem and major challenge to the global drug discovery programmes. Most of the genetic determinants that confer resistance to antibiotics are located on R-plasmids in bacteria. The present investigation was undertaken to investigate the ability of organic extract of the fruits of Helicteres isora to cure R-plasmids from certain clinical isolates. mMethods: Active fractions demonstrating antibacterial and antiplasmid activities were isolated from the acetone extracts of shade dried fruits of H. isora by bioassay guided fractionation. Minimal inhibitory concentration (MIC) of antibiotics and organic extracts was determined by agar dilution method. Plasmid curing activity of organic fractions was determined by evaluating the ability of bacterial colonies (pre treated with organic fraction for 18 h) to grow in the presence of antibiotics. The physical loss of plasmid DNA in the cured derivatives was further confirmed by agarose gel electrophoresis. Results: The active fraction did not inhibit the growth of either the clinical isolates or the strains harbouring reference plasmids even at a concentration of 400 mu g/ml. However, the same fraction could cure plasmids from Enterococcus faecalis, Escherichia coli, Bacillus cereus and E. coli (RP4) at curing efficiencies of 14, 26, 22 and 2 per cent respectively. The active fraction mediated plasmid curing resulted in the subsequent loss of antibiotic resistance encoded in the plasmids as revealed by antibiotic resistance profile of cured strains. The physical loss of plasmid was also confirmed by agarose gel electrophoresis. Interpretation & conclusions: The active fraction of acetone extract of H. isora fruits cured R-plasmids from Gram-positive and Gram-negative clinical isolates as well as reference strains. Such plasmid loss reversed the multiple antibiotic resistance in cured derivatives making them sensitive to low concentrations of antibiotics. Acetone fractions of H. isora may be a source to develop antiplasmid agents of natural origin to contain the development and spread of plasmid borne multiple antibiotic resistance.

Role of the environment in the interaction of nonintercalators with Z-DNA

Interaction of the DNA binding nonintercalators Netropsin, Distamycin and the mPD derivative with Z-DNA has been studied. It has been found that environmental factors like the solvent and added cations significantly modulate the interaction of these ligands with Z-DNA. However no definite Z to B transition in presence of these ligands was found in any case, in contrast to previously reported results (Ch. Zimmer, C. Marck and W. Guschlbauer, FEBS Lett. 154, 156-160 (1983)).

Transfer of saline tolerance from one strain of rice to another by injection of DNA

Direct injection of genomic DNA from salt tolerant cv. Pokkali into developing floral tillers on IR20 produced transgenic seeds similar to Pokkali in husk colour and which germinated well in 0.2 M NaCl and had a 4-6-fold higher proline content.

DNA duplex with the potential to change handedness after every half a turn

Polymorphic forms of the DNA duplex with long stretches of structural monotony are known. Several alternating purine-pyrimidine sequences have been shown to adopt left-handed Z-conformation. We report a DNA sequence d(CGCGCGATCGAT)n exhibiting alternating right-handed B and left-handed Z helical conformation after every half a turn. Further, this unusual conformation with change in handedness after every six base pairs was induced at physiological superhelical density.

Symmetrical Bisbenzimidazoles with Benzenediyl Spacer: The Role of the Shape of the Ligand on the Stabilization and Structural Alterations in Telomeric G-Quadruplex DNA and Telomerase Inhibition

The extremities of chromosomes end in a G-rich single-stranded overhang that has been implicated in the onset of the replicate senescence. The repeated sequence forming a G-overhang is able to adopt a four-stranded DNA structure called G-quadruplex, which is a poor substrate for the enzyme telomerase. Small molecule based ligands that selectively stabilize the telomeric G-quadruplex DNA, induce telomere shortening eventually leading to cell death. Herein, we have investigated the G-quadruplex DNA interaction with two isomeric bisbenzimidazole-based compounds that differ in terms of shape (V-shaped angular vs linear).While the linear isomer induced some stabilization of the intramolecular G-quadruplex structure generated in the presence of Na+ the other, having V-shaped central planar core, caused a dramatic structural alteration of the latter, above a threshold concentration. This transition was evident from the pronounced changes observed in the circular dichroism spectra and from the get mobility shift assa involving the G-quadruples DNA. Notably, this angular isomer could also induce the G-quadruplex formation in the absence of any added cation. The ligand-quadruples complexes were investigated by computational molecular modeling, providing further information on structure-activity relationships. Finally, TRAP (telomerase repeat amplification protocol) experiments demonstrated that the angular isomer is selective toward the inhibition of telomerase activity.

Photocytotoxicity and DNA cleavage activity of L-arg and L-lys Schiff base oxovanadium(IV) complexes having phenanthroline bases

Oxovanadium(IV) complexes [VO(sal-argH)(B)] Cl (1-3) and [VO(sal-lysH)(B)] Cl (4-6), where sal-argH2 and sal-lysH(2) are N-salicylidene-L-arginine and N-salicylidene-L-lysine Schiff bases and B is a phenanthroline base, viz. 1,10-phenanthroline (phen in 1 and 4); dipyrido[3,2-d: 2', 3'-f] quinoxaline (dpq in 2 and 5) and dipyrido[3,2-a: 2', 3'-c] phenazine (dppz in 3 and 6), have been prepared, characterized and their DNA photocleavage activity studied. Complex 1, characterized by X-ray crystallography, shows the presence of a vanadyl group in VIVO3N3 coordination geometry with a tridentate Schiff base having a pendant guanidinium moiety and bidentate phen ligand. The complexes exhibit a d-d band at similar to 715 nm in 20% DMF-Tris-HCl buffer. The complexes are redox active showing cathodic and anodic responses near -1.0 V and 0.85 V (vs. SCE) for the V(IV)-V(III) and V(V)-V(IV) couples, respectively, in DMF-Tris-HCl buffer. The complexes bind to calf thymus DNA giving Kb values in the range of 3.8 x 10(4) to 1.6 x 10(5) M-1. Thermal denaturation and viscosity data suggest DNA groove binding nature of the complexes. The complexes do not show any `chemical nuclease'' activity in dark in the presence of 3-mercaptopropionic acid or H2O2. The dpq and dppz complexes are efficient photocleavers of plasmid DNA in UV-A (365 nm) and red light (676 nm) via singlet oxygen pathway. The dppz complexes exhibit photocytotoxicity in HeLa cancer cells giving IC50 values of 15.4 mu M for 3 and 17.5 mu M for 6 in visible light while being non-toxic in dark giving IC50 values of > 100 mu M.

Studies on hypomethylation of liver DNA during early stages of chemical carcinogenesis in rat liver

Our finding that the inhibitors of DNA methylation, 5-azacytidine, 5-azadeoxycytidine or adenosine dialdehyde, given after a carcinogen all potentiated initiation suggested that hypomethylation of DNA during repair synthesis of DNA might play a role in the initiation of the carcinogenic process. To examine this aspect further, we have asked the question, do the nodules which develop from initiated cells after promotion with 1% orotic acid exhibit an altered methylation pattern in their DNA? The methylation status of the DNA from nodules has been examined using the restriction endonucleases HpaII/MspI and HhaI which distinguish between methylated and unmethylated cytosines in their nucleotide recognition DNA 5'-CCGG and 5'-GCGC respectively. The proto-oncogenes, c-myc, c-fos and c-Ha-ras, in the DNA were primarily studied in this investigation because of their possible involvement in cell proliferation and/or in cell transformation and tumorigenesis. The results indicate that in the nodule DNA, c-myc and c-fos are hypomethylated in the sequence of CCGG while the c-Ha-ras shows hypomethylation in the alternating GCGC sequence. This methylation pattern seen in the nodule DNA is not found in the DNA of the non-nodular surrounding liver or liver tissue after exposure to promoter or carcinogen alone. It is also not found in the DNA of regenerating liver. It is particularly significant that the methylation patterns in the c-myc and c-Ha-ras regions are similar to those found in several cancer tissues. The results suggest that this methylation pattern is acquired early in the carcinogenic process and raises the question whether it has any bearing on the process.

sRNAscanner: A Computational Tool for Intergenic Small RNA Detection in Bacterial Genomes

Background:Bacterial non-coding small RNAs (sRNAs) have attracted considerable attention due to their ubiquitous nature and contribution to numerous cellular processes including survival, adaptation and pathogenesis. Existing computational approaches for identifying bacterial sRNAs demonstrate varying levels of success and there remains considerable room for improvement. Methodology/Principal Findings: Here we have proposed a transcriptional signal-based computational method to identify intergenic sRNA transcriptional units (TUs) in completely sequenced bacterial genomes. Our sRNAscanner tool uses position weight matrices derived from experimentally defined E. coli K-12 MG1655 sRNA promoter and rho-independent terminator signals to identify intergenic sRNA TUs through sliding window based genome scans. Analysis of genomes representative of twelve species suggested that sRNAscanner demonstrated equivalent sensitivity to sRNAPredict2, the best performing bioinformatics tool available presently. However, each algorithm yielded substantial numbers of known and uncharacterized hits that were unique to one or the other tool only. sRNAscanner identified 118 novel putative intergenic sRNA genes in Salmonella enterica Typhimurium LT2, none of which were flagged by sRNAPredict2. Candidate sRNA locations were compared with available deep sequencing libraries derived from Hfq-co-immunoprecipitated RNA purified from a second Typhimurium strain (Sittka et al. (2008) PLoS Genetics 4: e1000163). Sixteen potential novel sRNAs computationally predicted and detected in deep sequencing libraries were selected for experimental validation by Northern analysis using total RNA isolated from bacteria grown under eleven different growth conditions. RNA bands of expected sizes were detected in Northern blots for six of the examined candidates. Furthermore, the 5'-ends of these six Northern-supported sRNA candidates were successfully mapped using 5'-RACE analysis. Conclusions/Significance: We have developed, computationally examined and experimentally validated the sRNAscanner algorithm. Data derived from this study has successfully identified six novel S. Typhimurium sRNA genes. In addition, the computational specificity analysis we have undertaken suggests that similar to 40% of sRNAscanner hits with high cumulative sum of scores represent genuine, undiscovered sRNA genes. Collectively, these data strongly support the utility of sRNAscanner and offer a glimpse of its potential to reveal large numbers of sRNA genes that have to date defied identification. sRNAscanner is available from: http://bicmku.in:8081/sRNAscanner or http://cluster.physics.iisc.ernet.in/sRNAscanner/.

Antibodies raised against denatured DNA bind to double-stranded DNA

Antibodies raised against denatured DNA complexed with methylated bovine serum albumin have been reported to react with ssDNA but not with dsDNA. Using a highly sensitive avidin-biotin microELISA, we report that such antibodies also bind to dsDNA. Antibodies which reacted with ssDNA and dsDNA were found to be IgG type. The antibodies did not react with tRNA and rRNA. The binding of antibodies to dsDNA was partially inhibited dy individual deoxyribonucleotides. ssDNA as well as dsDNA inhibited the binding of antibodies to dsDNA. The binding of these antibodies to supercoiled and relaxed forms of pBR322 DNA was demonstrated by gel retardation assay. The cross-reaction with ssDNA was observed even after affinity purification on native DNA-cellulose. The antibodies were also shown to bind to poly(dA-dT)·poly(dA-dT)

A self-consistent formulation for analysis and generation of non-uniform DNA structure

A fully self-consistent formulation is described here for the analysis and generation of base-pairs in non-uniform DNA structures, in terms of various local parameters. It is shown that the internal "wedge parameters" are mathematically related to the parameters describing the base-pair orientation with respect to an external helix axis. Hence any one set of three translation and three rotation parameters are necessary and sufficient to completely describe the relative orientation of the base-pairs comprising a step (or doublet). A general procedure is outlined for obtaining an average or global helix axis from the local helix axes for each step. A graphical representation of the local helix axes in the form of a polar plot is also shown and its application for estimating the curvature of oligonucleotide structures is illustrated, with examples of both A and B type structures.

A general procedure for generation of curved DNA molecules.

A general method for generation of base-pairs in a curved DNA structure, for any prescribed values of helical parameters--unit rise (h), unit twist (theta), wedge roll (theta R) and wedge tilt (theta T), propeller twist (theta p) and displacement (D) is described. Its application for generation of uniform as well curved structures is also illustrated with some representative examples. An interesting relationship is observed between helical twist (theta), base-pair parameters theta x, theta y and the wedge parameters theta R, theta T, which has important consequences for the description and estimation of DNA curvature.