Characterization of the DNA-binding domain of beta protein, a component of phage lambda Red-pathway by UV catalyzed cross-linking

beta protein, a key component of Red-pathway of phage lambda is necessary for its growth and general genetic recombination in recombination-deficient mutants of Escherichia coli. To facilitate studies on structure-function relationships, we overexpressed beta protein and purified it to homogeneity. A chemical cross-linking reagent, glutaraldehyde, was used to stabilize the physical association of beta protein in solution. A 67-kDa band, corresponding to homodimer, was identified after separation by SDS-polyacrylamide gel electrophoresis. Stoichiometric measurements indicated a site-size of 1 monomer of beta protein/5 nucleotide residues. Electrophoretic gel mobility shift assays suggested that beta protein formed stable nucleoprotein complexes with 36-mer, but not with 27- or 17-mer DNA. Interestingly, the interaction of beta protein with DNA and the stability of nucleoprotein complexes was dependent on the presence of MgCl2, and the binding was abolished by 250 mM NaCl. The K-d of beta protein binding to 36-mer DNA was on the order of 1.8 x 10(-6) M. Photochemical cross-linking of native beta protein or its fragments, generated by chymotrypsin, to 36-mer DNA was performed to identify its DNA-binding domain. Characterization of the cross-linked peptide disclosed that amino acids required for DNA-binding specificity resided within a 20-kDa peptide at the N-terminal end. These findings provide a basis for further understanding oi the structure and function of beta protein.

DNA cleavage by intercalatable cobalt-bispicolylamine complexes activated by visible light

Two intercalatable Co-II-complexes of anthryl or anthraquinone attached bispicolylamine derivatives cleave plasmid pTZ19R DNA spontaneously upon exposure to visible light under ambient conditions.

Chain folding and A:T pairing in human telomeric DNA: a model-building and molecular dynamics study

The various types of chain folding and possible intraloop as well as interloop base pairing in human telomeric DNA containing d(TTAG(3)) repeats have been investigated by model-building, molecular mechanics, and molecular dynamics techniques. Model-building and molecular mechanics studies indicate that it is possible to build a variety of energetically favorable folded-back structures with the two TTA loops on same side and the 5' end thymines in the two loops forming TATA tetrads involving a number of different intraloop as well as interloop A:T pairing schemes. In these folded-back structures, although both intraloop and interloop Watson-Crick pairing is feasible, no structure is possible with interloop Hoogsteen pairing. MD studies of representative structures indicate that the guanine-tetraplex stem is very rigid and, while the loop regions are relatively much more flexible, most of the hydrogen bonds remain intact throughout the 350-ps in vacuo simulation. The various possible TTA loop structures, although they are energetically similar, have characteristic inter proton distances, which could give rise to unique cross-peaks in two-dimensional nuclear Overhauser effect spectroscopy (NOESY) experiments. These folded-back structures with A:T pairings in the loop region help in rationalizing the data from chemical probing and other biochemical studies on human telomeric DNA.

Characterization of a Negative cis-Acting DNA Element Regulating the Transcription of CYP2B1/B2 Gene in Rat Liver

The region -160 to -127 nt of the upstream of CYP-2B1/B2 gene has been found to function as a negative cis-acting element on the basis of DNase-I footprint and gel mobility shift assays as well as cell-free transcriptional assays using Bal-31 mutants. A reciprocal relationship in the interaction of the negative and the recently characterized positive elements with their respective protein factors has been found under repressed and induced conditions of the gene. The negative element also harbors the core glucocorticoid responsive sequence, TGTCCT. It is concluded that the negative element mediates the repressed state of the gene under the uninduced condition and also mediates the repressive effect of dexamethasone, when given along with the inducer phenobarbitone in rats. Dexamethasone is able to antagonize the effects of phenobarbitone at as low a concentration as 100 mu g/kg body wt in these animals. (C) 1995 Academic Press,Inc.

Functional analysis of conserved motifs in EcoP15I DNA methyltransferase

EcoP15I DNA methyltransferase recognizes the sequence 5'-CAGCAG-3' and transfers a methyl group to N-6 of the second adenine residue in the recognition sequence. All N-6 adenine methyltransferases contain two highly conserved sequences, FxGxG (motif I), postulated to form part of the S-adenosyl-L-methionine binding site and (D/N/S)PP(Y/F) (motif IV) involved in catalysis. We have altered the second glycine residue in motif I to arginine and serine, and substituted tyrosine in motif IV with tryptophan in EcoP15I DNA methyltransferase, using site-directed mutagenesis. The mutant enzymes were overexpressed, purified and characterized by biochemical methods. The mutations in motif I completely abolished AdoMet binding but left target DNA recognition unaltered. Although the mutation in motif IV resulted in loss of enzyme activity, we observed enhanced crosslinking of S-adenosyl-L-methionine and DNA. This implies that DNA and AdoMet binding sites are close to motif IV. Taken together, these results reinforce the importance of motif I in AdoMet binding and motif IV in catalysis. Additionally, limited proteolysis and UV crosslinking experiments with EcoP15I DNA methyltransferase imply that DNA binds in a cleft formed by two domains in the protein. Methylation protection analysis provides evidence for the fact that EcoP15I DNA MTase makes contacts in the major groove of its substrate DNA. Interestingly, hypermethylation of the guanine residue next to the target adenine residue indicates that the protein probably flips out the target adenine residue. (C) 1996 Academic Press Limited

Quantitation of spermatogenesis by DNA flow cytometry: Comparative study among six species of mammals

Suspensions of testicular germ cells from six species of mammals were prepared and stained for the DNA content with a fluorochrome (ethidium bromide) adopting a common technique and subjected to DNA flow cytometry. While uniform staining of the germ cells of the mouse, hamster, rat and monkey could be obtained by treating with 0.5% pepsin for 60 min followed by staining with ethidium bromide for 30 min, that of the guinea pig and rabbit required for optimal staining pepsinization for 90 min and treatment with ethidium bromide for 60 min. The procedure adopted here provided a uniform recovery of over 80% of germ cells with each one of the species tested and the cell population distributed itself according to the DNA content (expressed as C values) into 5 major classes-spermatogonia (2C), cells in S-phase, primary spermatocytes (4C), round spermatids (1C), and elongating/elongated spermatids (HC). Comparison of the DNA distribution pattern of the germ cell populations between species revealed little variation in the relative quantities of cells with 2C (8-11%), S-phase (6-9%), and 4C (6-9%) amount of DNA. Though the spermatid cell populations exhibited variations (1C:31-46%, HCI:7-20% and and HC2:11-25%) they represented the bulk of germ cells (70-80%). The overall conversion of 2C to 1C (1C:2C ratio) and meiotic transformation of 4C cells to IC (1C:4C ratio) kinetics were relatively constant between the species studied. The present study clearly demonstrates that DNA flow cytometry can be adopted with ease and assurance to quantify germ cell transformation and as such spermatogenesis by analysing a large number of samples with consistency both within and across the species barrier. Any variation from the norms in germ cell proportions observed following treatment, for e.g. hormonal stimulation or deprivation can then be ascribed due to a specific effect of the hormone/drug on single/multiple steps in germ cell transformation

Development of specific DNA probes and their usage in the detection of Plasmodium vivax infection in blood

The application of nucleic acid probes, in the detection of pathogenic micro-organisms, has become an integral part of diagnostic technologies. In this study, Plasmodium vivax-specific DNA probes have been identified by carrying out genomic subtractive hybridization. In this approach, the recombinant clones from a P. vivax genomic library are screened with radiolabelled human and P. falciparum DNA. The colonies which react with labelled P. falciparum and human DNA are eliminated and those which do not produce any autoradiographic signal have been subjected to further screening procedures. Three Fl vivax specific DNA probes have been obtained by these repeated screenings. Further analyses indicate that these probes are specific and sensitive enough to detect P. vivax infection in clinical blood samples when used in a non-radioactive DNA hybridization assay. (C) 1995 Academic Press Limited

Photocytotoxicity and DNA photocleavage activity of La(III) and Gd(III) complexes of phenanthroline bases

Lanthanide(II) complexes La(B)(acac)(3)] (1-3) and Gd(B)(acac)(3)] (4-6), where B is a N,N-donor phenanthroline base, viz., 1,10-phenanthroline (phen in 1, 4), dipyrido3,2-d:2',3'-f]quinoxaline (dpq in 2, 5) and dipyrido3,2-a:2',3'-c]phenazine (dppz in 3, 6), have been prepared and characterized. The Gd(111) complexes 4 6 are structurally characterized by single crystal X-ray crystallography. The complexes display GdO6N2 coordination with the ligands showing bidentate chelating mode of bonding. The complexes are non-electrolytic in aqueous DMF and exhibit ligand-centered absorption bands in the UV region. The dppz complexes show a band at 380 nm in DMF. The La(111) complexes are diamagnetic. The Gd(III) complexes are paramagnetic with magnetic moment that corresponds to seven unpaired electrons. The Complexes are avid binders to calf thymus DNA giving K-b values in the range of 4.7 x 10(4) 6.1 x 10(5) M-1 with a relative binding order: 3, 6 (dppz) > 2, 5 (dpq) > 1, 4 (phen). The binding data suggest DNA surface and/or groove binding nature of the complexes. The dpq and dppz complexes efficiently cleave SC DNA to its nicked circular form in UV-A light of 365 nm via formation of both singlet oxygen (O-1(2)) and hydroxyl radical (HO center dot) species. The dppz complexes 3 and 6 exhibit significant PDT effect in He La cervical cancer cells giving respective IC50 value of 460(+/- 50) and 530(+/- 30) nM in UV-A light of 365 rim, and are essentially non-toxic in dark with an IC50 value of >100 mu M. The dppz ligand alone is cytotoxic in dark and UV-A light. A significant decrease in the dark toxicity of the dppz base is observed on binding to the Ln(III) ion while retaining its photocytotoxicity.

DNA Condensation by the Rat Spermatidal Protein TP2 Shows GC-Rich Sequence Preference and Is Zinc Dependent

Transition protein-2 (TP2), isolated from rat testes, was recently shown to be a zinc metalloprotein. We have now carried out a detailed analysis of the DNA condensing properties of TP2 with various polynucleotides using circular dichroism spectroscopy. The condensation of the alternating copolymers by TP2 (incubated with 10 mu M ZnSO4), namely, poly(dG-dC). poly(dG-dC) and poly(dA-dT). poly(dA-dT), was severalfold higher than condensation of either of the homoduplexes poly(dG). poly-(dC) and poly(dA). poly(dT) or rat oligonucleosomal DNA. Between the two alternating copolymers, poly(dG-dC). poly(dG-dC) was condensed 3.2-fold more effectively than poly(dA-dT). poly(dA-dT). Preincubation of TP2 with 5 mM EDTA significantly reduced its DNA-condensing property. Interestingly, condensation of the alternating copolymer poly(dI-dC). poly(dI-dC) by TP2 was much less as compared to that of poly(dG-dC). poly(dG-dC). The V8 protease-derived N-terminal fragment (88 aa) condensed poly(dA-dT). poly(dA-dT) to a very small extent but did not have any effect on poly(dG-dC). poly-(dG-dC). The C-terminal fragment (28 aa) was able to condense poly(dA-dT) . poly(dA-dT) more effectively than poly(dG-dC). poly(dG-dC). These results suggest that TP2 in its zinc-coordinated form condenses GC-rich polynucleotides much more effectively than other types of polynucleotides. Neither the N-terminal two-thirds of TP2 which is the zinc-binding domain nor the C-terminal basic domain are as effective as intact TP2 in bringing about condensation of DNA.

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.

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.