DNA recognition by the EcoP15I and EcoPI modification methyltransferases

The DNA-binding properties of the EcoP15I DNA methyltransferase (M . EcoP15I; MTase) were studied using electrophoretic mobility shift assays. We show by molecular size-exclusion chromatography and dimethyl suberimidate crosslinking that M . EcoP15I is a dimer in solution. While M . EcoP15I binds approx. threefold more tightly to its recognition sequence, 5'-CAGCAG-3', than to non-specific sequences in the presence of AdoMet or its analogs, the discrimination between specific and non-specific sequences significantly increases in presence of ATP. These results suggest for the first time a role for ATP in DNA recognition by type-III restriction-modification enzymes. Furthermore, we show that although c2 EcoPI mutant MTases are defective in AdoMet binding, they are still able to bind DNA in a sequence-specific manner.

Isolation of an 800 kb contiguous DNA fragment encompassing a 3.5-cM region of chromosome 1 in Arabidopsis using YAC clones

The apetalal mutation of Arabidopsis affects floral meristem identity and the development of sepal and petal primordia of the flower. We mapped the available RFLP markers on chromosome 1 that are in the general vicinity of apetalal on a fine structure map and then chose the closest RFLP as a starting point for contiguous DNA (contig) generation. We report here a contig of about 800 kilobases (kb) that spans a 3.5 cM region of chromosome 1. We used genomic libraries of Arabidopsis prepared in yeast artificial chromosome (YAC) vectors and the detailed characterization of 19 YACs is reported. RFLPs displayed by the end fragments from the walk were mapped to align and correlate the genetic and physical maps for this region of chromosome 1. In this segment of the genome, 1 cM corresponds to a little over 200 kb of physical distance.

DNA binding properties of some cationic acridine derivatives

Four cationic acridine derivatives have been synthesized. The positively charged amine residue in one of these is connected directly on to the acridine nucleus and in three other acridines, the amines are connected via a 9-CH2 unit to acridine. We have investigated the binding of these acridines with mammalian DNA by absorption titration, UV- and induced-CD spectroscopy and competitive ethidium bromide displacement fluorescence assay. The effects on the DNA duplex denaturation melting temperatures upon binding of each one of these are also examined. The results obtained herein clearly show that the introduction of a -CH2 group in the im mediate vicinity of the interrelation moiety introduces alterations in the DNA binding characteristics of the resulting acridines.

Purification and characterization of DNA-dependent RNA polymerase II from Candida utilis

DNA-dependent RNA polymerase II from Candida utilis has been purified to near homogeneity. The purified enzyme resolved into three subforms, viz. IIO, IIA and IIB. On SDS-PAGE the enzyme showed ten polypeptides with molecular weights in the range of 205 kDa to 14 kDa. By two dimensional electrophoresis (IEF followed by SDS-PAGE) the presence of basic and acidic polypeptides has been demonstrated. The enzyme showed Km values of 5, 5.6 and 8 mu M for GTP, CTP and ATP, respectively, and the activity was inhibited by low levels of oc-amanitin and antibodies raised against bovine RNA polymerase II. By Western blot analysis the enzyme was found to cross-react with antibodies to bovine RNA polymerase II. RNA polymerase II from G. utilis is a phosphoprotein, the subunits RPB1 and RPB10 were found to be phosphorylated. Analysis of carboxy-terminal domain indicated that it was functionally redundant at least in case of nonspecific transcription, implicating its role in other nuclear processes, such as promoter specific initiation or transcription activation or RNA processing.

Schiff base oxovanadium(IV) complexes of phenanthroline bases showing DNA photocleavage activity at near-IR light and photocytotoxicity

Oxovanadium(IV) complexes VO(L)(B)](ClO4) (1-3) of N-2-pyridylmethylidine-2-hydroxyphenylamine (HL) Schiff base and phenanthroline bases (B), viz. 1,10-phenanthroline (phen in 1), dipyrido3,2-d: 2',3'-f] quinoxaline (dpq in 2) or dipyrido3,2-a: 2',3'-c] phenazine (dppz in 3), were prepared, characterized and their DNA binding property, photo-induced DNA cleavage activity and photocytotoxicity in HeLa cells studied. The crystal structure of 1 shows the presence of a VO2+ moiety in VO2N4 coordination geometry. The complexes show a d-d band at similar to 830 nm in DMF. The complexes display an oxidative V(V)-V(IV) response near 0.5 V versus SCE and a reductive V(IV)/V(III) response near -0.65 V in DMF -0.1 M TBAP. The complexes that are avid binders to CT DNA giving K-b values within 7.1 x 10(4) to 3.2 x 10(5) M-1, do not show any significant chemical nuclease activity in presence of 3-mercaptopropionic acid or glutathione. The dpq and dppz complexes are photocleavers of pUC19 DNA in UV-A light of 365 nm forming both O-1(2) and (OH)-O-center dot radicals and in near-IR light of 785 nm forming (OH)-O-center dot radicals. The dppz complex exhibits photocytotoxicity in visible light in HeLa cells (IC50 = 6.8 mu M). Flow-cytometric study on this complex shows a high sub-G1 phase in light compared to dark indicating PDT effect. (C) 2011 Elsevier B. V. All rights reserved.

Preferential condensation of SAR-DNA by histone H1 and its SPKK containing octapeptide repeat motif

Linker histone H1 binds preferentially the scaffold associated region (SAR) DNA elements that contain characteristic oligo dA . dT tracts. In the present study, we have compared the condensation brought about by histone H1 of a SAR DNA fragment in the histone spacer region of Drosophila melanogaster with that of a random DNA (pBR322 EcoRI-SalI) fragment by circular dichroism spectroscopy. The condensation of the SAR DNA fragment by histone H1 is 3-4-fold higher than that of the random DNA fragment. A 16-mer peptide, ATPKKSTKKTPKKAKK, the sequence that is present in the C-terminus of histone H1d, which has recently been shown to possess DIVA and chromatin condensing properties, also condenses the SAR DNA fragment preferentially in a highly cooperative manner. We have proposed a model for the dynamics of chromatin structure involving histone H1-SAR DNA interaction through SPKK containing peptide motifs and its competition by AT-hook peptides present in the nonhistone chromosomal proteins like HMG-I and HMG-Y.

Role of hydrophobic effect and surface charge in surfactant-DNA association

Ethidium bromide is one of the best known DNA intercalator. Upon intercalation inside DNA, the fluorescence due to ethidium bromide gets enhanced by many orders of magnitude. In this paper, we employed ethidium bromide as a probe for studying surfactant-DNA complexation using fluorescence spectroscopy and agarose gel electrophoresis. Surfactants of different charge types and chain lengths were used and the results were compared with that of the related small organic cations or salts under comparable conditions. The cationic surfactants induced destabilization of the ethidium bromide-DNA complex at concentrations in orders of magnitude lower than that of the small organic cations or salts. In contrast however, the anionic surfactants failed to promote any such destabilization of probe-DNA complex. DNA loses its ethidium bromide stainability in the presence of high concentration of cationic surfactant aggregates as revealed from agarose gel electrophoresis experiments. Inclusion of surfactants and other additives into the DNA generally enhanced the DNA double-strand to single strand transition melting temperatures by a few degrees, in a concentration-dependent manner and at high surfactant concentration melting profiles got broadened.

Is O-1(2) alone sufficient for DNA cleavage? Possible involvement of paramagnetic intermediates

It is proposed that singlet dioxygen reacting with guanosine or deoxyguanosine part of nucleotides does not, by itself, cause DNA cleavage. The strand break originates at the endoperoxide stage whenever this link evolves into a O-centered radical. The O-centered radical is then in a good spatial position to abstract an hydrogen intramolecularly from the ribose or desoxyribose part of the nucleotide. The carbon centered radical thus formed on the sugar part may lead to strand break either by a p-scission mechanism or by an homolytically induced solvolysis. High pH could also induce cleavage after the endoperoxide stage via a base catalyzed ring chain protomerism.

Sex-specific organisation of middle repetitive DNA sequences in the mealybug Planococcus lilacinus

Differential organisation of homologous chromosomes is related to both sex determination and genomic imprinting in coccid insects, the mealybugs. We report here the identification of two middle repetitive sequences that are differentially organised between the two sexes and also within the same diploid nucleus. These two sequences form a part of the male-specific nuclease-resistant chromatin (NRC) fraction of a mealybug Planococcus lilacinus. To understand the phenomenon of differential organisation we have analysed the components of NRC by cloning the DNA sequences present, deciphering their primary sequence, nucleosomal organisation, genomic distribution and cytological localisation, Our observations suggest that the middle repetitive sequences within NRC are functionally significant and we discuss their probable involvement in male-specific chromatin organisation.

Structural characterisation of a uracil containing hairpin DNA by NMR and molecular dynamics

Three-dimensional (3D) structure of a hairpin DNA d-CTAGAGGATCCTTTUGGATCCT (22mer; abbreviated as U4-hairpin), which has a uracil nucleotide unit at the fourth position from the 5' end of the tetra-loop has been solved by NMR spectroscopy. The H-1 resonances of this hairpin have been assigned almost completely. NMR restrained molecular dynamics and energy minimisation procedures have been used to describe the 3D structure of the U4 hairpin. This study establishes that the stem of the hairpin adopts a right handed B-DNA conformation while the T-12 and U-15 nucleotide stack upon 3' and 5' ends of the stem, respectively. Further, T-14 stacks upon both T-12 and U-15 while T-13 partially stacks upon T-14. Very weak stacking interaction is observed between T-13 and T-12. All the individual nucleotide bases adopt 'anti' conformation with respect to their sugar moiety. The turning phosphate in the loop is located between T-13 and T-14. The stereochemistry of U-15 mimics the situation where uracil would stack in a B-DNA conformation. This could be the reason as to why the U4-hairpin is found to be the best substrate for its interaction with uracil DNA glycosylase (UDG) compared to the other substrates in which the uracil is at the first, second and third positions of the tetra-loop from its 5' end, as reported previously.

DNA binding properties of novel dansylated distamycin analogues in which the fluorophore is directly conjugated to the N-methyl-pyrrole carboxamide backbone

Polyamides that are structural analogues of the naturally occurring DNA minor groove binding antibiotic distamycin (Dst) are promising candidates as gene modulators. Developing strategies for the large scale screening and monitoring of the cellular distribution of such ligands would aid the faster discovery of molecules, which would have eventual utility in molecular biology and medicine. Attachment of fluorescent tags would be a useful step towards this end. A fundamental question in this connection is whether the tag modifies the DNA binding affinity of the parent compounds. Towards answering this question, we have developed two oligopeptides that bear the dansyl (N, N-dimethylaminonaphthalene sulfonamido fluorophore) coupled directly to the N-terminus of the conjugated N-methylpyrrole carboxamide network, and possess three or four N-methyl pyrrole carboxamide units (abbreviated as Dn3 and Dn4 respectively). DNA binding abilities of these molecules were assessed from fluorescence titration experiments, duplex-DNA T-m analysis (employing both UV and fluorescence spectroscopy), induced circular dichroism measurements (ICD), salt dependence of ICD and apparent binding constant measurements (K-app) employing ethidium bromide (EtBr) displacement assay. Both these molecules 'reported' DNA binding in the form of an enhanced fluorescence emission. As judged from the ICD measurements, salt dependence of ICD, T-m analysis and K-app measurements, the binding affinities of the molecules that possessed dansyl group at their N-termini were lower than the ones with equivalent number of amide units, but possessed N-methylpyrrole carboxamide unit at their N-termini. These results would have implications in the future design of fluorescent polyamides.

DNA topoisomerase I from mycobacteria - A potential drug target

DNA topoisomerases are ubiquitous group of enzymes altering the topology of DNA by concerted breakage and rejoining of the phosphodiester backbone of DNA. The enzymes are classified based on the pattern of DNA cleavage. Type IA enzymes found in all bacteria nick the DNA and attach themselves covalently to the 5' side of the nick during the first transesterification reaction. Most of the information on this group of enzymes comes from studies with E. coli topoisomerase I and III. Members of type IA group are single subunit Zn++ metalloenzymes recognizing single stranded DNA without high degree of sequence specificity during relaxation reaction of negatively super coiled DNA. So far no inhibitors are known for this group of enzymes inspite of their important role in maintaining homeostasis of DNA topology. Molecular characterization of DNA topoisomerase I from mycobacteria has revealed some of the important features of type IA enzymes hitherto unknown and provide scope for identifying novel inhibitors. The present review describes the recent developments in the area summarizing the distinctive features of mycobacterial topoisomerase I. The enzyme has several properties not shared by either type IA or 113 enzymes with respect to DNA binding, recognition, sequence specificity and interaction pattern. The physiological basis of the unusual features is discussed. The unique properties described would aid in developing the enzyme as a target molecule in pharmaceutical design. In addition, the findings lead to address some fundamental questions on the intracellular role of topoisomerase I in the biology of mycobacteria which are one of the most formidable group of pathogenic organisms.

DNA Free Energy-Based Promoter Prediction and Comparative Analysis of Arabidopsis and Rice Genomes

The cis-regulatory regions on DNA serve as binding sites for proteins such as transcription factors and RNA polymerase. The combinatorial interaction of these proteins plays a crucial role in transcription initiation, which is an important point of control in the regulation of gene expression. We present here an analysis of the performance of an in silico method for predicting cis-regulatory regions in the plant genomes of Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) on the basis of free energy of DNA melting. For protein-coding genes, we achieve recall and precision of 96% and 42% for Arabidopsis and 97% and 31% for rice, respectively. For noncoding RNA genes, the program gives recall and precision of 94% and 75% for Arabidopsis and 95% and 90% for rice, respectively. Moreover, 96% of the false-positive predictions were located in noncoding regions of primary transcripts, out of which 20% were found in the first intron alone, indicating possible regulatory roles. The predictions for orthologous genes from the two genomes showed a good correlation with respect to prediction scores and promoter organization. Comparison of our results with an existing program for promoter prediction in plant genomes indicates that our method shows improved prediction capability.

GyrI: a counter-defensive strategy against proteinaceous inhibitors of DNA gyrase

DNA gyrase is the target of two plasmid-encoded toxins CcdB and microcin B17, which ensure plasmid maintenance. These proteins stabilize gyrase-DNA covalent complexes leading to double-strand breaks in the genome. In contrast, the physiological role of chromosomally encoded inhibitor of DNA gyrase (Gyrl) in Escherichia coli is unclear and its mechanism of inhibition has not been established. We demonstrate that the mode of inhibition of GyrI is distinct from all other gyrase inhibitors. It inhibits DNA gyrase prior to, or at the step of, binding of DNA by the enzyme. Gyrl reduces intrinsic as well as toxin-stabilized gyrase-DNA covalent complexes. Furthermore, Gyri reduces microcin B17-mediated double-strand breaks in vivo, imparting protection to the cells against the toxin, substantiating the in vitro results. Thus, Gyrl is an antidote to DNA gyrase-specific proteinaceous poisons encoded by plasmid addiction systems.