Substrate specificity of Ca2+,Mg2+-dependent DNAase from sea urchin (Strongylocentrotus intermedius) embryos

Ca2+,Mg2+-dependent DNAse from sea urchin embryos is specific to the secondary structure of substrates irrespective of the nature of activating cations. The enzyme does not split synthetic single-stranded oligo and polynucleotides, such as d(pTpTpTpCpC), d(pGpGpTpTpT). d(pApApTpTpC), d(pGpApApTpTpC), d(pA)5-poly(dT), d(pApApTpTpC)-poly(dT), poly(dA) and poly (dT) and hydrolyses the double-stranded substrates poly d(AT), poly (dA) . poly (dT) and highly polymerized DNA. Native double-stranded DNA from salmon and phage T7 is split by the enzyme at a higher rate than that of denaturated DNA of salmon and single-stranded DNA of phage M13. The high rate of poly(dA) . poly(dT) and poly d(AT) hydrolysis and the stability of poly(dG) . poly(dC) to the effect of the enzyme suggest a certain specificity of the enzyme to the nature of nitrogenous bases at the hydrolyzed phosphodiester bond of the substrate.     

Models of triple-stranded polynucleotides with optimised stereochemistry.

Detailed models are presented for the triple-stranded polynucleotide helices of poly (U)-poly (A)-poly (U) (two forms), poly (U)-poly d (A) -poly (U), poly d(C)-poly d(I)-poly d(C), poly d(T)-polyd(A)-poly d(T) and poly (I)-poly (A)-poly (I). The models were genrated using a computerized, linked-atom procedure which preserves standard bond lengths, bond anglesand sugar ring conformations, constrains the helices to have the pitches and symmetries observed in X-ray diffraction experiments, and optimises the non-bonded interatomic contacts including hydrogen bonds. The possible biological sigificance of such complexes is discussed.     

Differences in melting behavior between homopolymers and copolymers of DNA: Role of nonbonded forces for GC and the role of the hydration spine and premelting transition for AT

Melting measurements of the mono-base-pair DNA polymers showed that the melting temperature T_m of the B-DNA homopolymer poly (dA ) · poly (dT) is higher than that of the copolymer poly [d(A-T)]. On the other hand, the T_mof the B-DNA homopolymer poly (dG) · poly (dC) is lower than that of the copolymer poly [d (G-C)]. From a structural point of view, the cross-strand base-stacking interaction in a DNA homopolymer is weaker than that in a DNA copolymer with the same base pair. One would then expect that all the DNA homopolymers are less stable than the copolymer with the same base pair. We find that the inversion of the melting order seen in the AT mono-base-pair DNA polymers is caused by the enhanced thermal stability of poly (dA) · poly (dT) from a well-defined spine of hydration attached to its minor groove. In this paper we employ the modified self-consistent phonon theory to calculate base-pair opening probabilities of four B-DNA polymers: poly(dA)-poly(dT), poly(dG) · poly(dC), poly[d(A-T)], and poly[d(G-C)] at temperatures from room temperature through the melting regions. Our calculations show that the spine of hydration can give the inverted melting order of the AT polymers as compared to the GC polymers in fair agreement with experimental measurements. Our calculated hydration spine disruption behavior in poly(dA) · poly(dT) at premelting temperatures is also in agreement with experimentally observed premelting transitions in poly (dA) · poly (dT). The work is in a sense a test of the validity of our models of nonbonded interactions and spine of hydration interactions. We find we have to develop the concept of a strained bond to fit observations in poly (dA) · poly(dT). The strained-bond concept also explains the otherwise anomalous stability of the hydration chain. © 1993 John Wiley & Sons, Inc.     

Microcalorimetric investigation of DNA, poly(dA)poly(dT) and poly[d(A-C)]poly[d(G-T)] melting in the presence of water soluble (meso tetra (4 N oxyethylpyridyl) porphyrin) and its Zn complex

Thermodynamic parameters of melting process (DeltaHm, Tm, DeltaTm) of calf thymus DNA, poly(dA)poly(dT) and poly(d(A-C)).poly(d(G-T)) were determined in the presence of various concentrations of TOEPyP(4) and its Zn complex. The investigated porphyrins caused serious stabilization of calf thymus DNA and poly poly(dA)poly(dT), but not poly(d(A-C))poly(d(G-T)). It was shown that TOEpyp(4) revealed GC specificity, it increased Tm of satellite fraction by 24 degrees C, but ZnTOEpyp(4), on the contrary, predominantly bound with AT-rich sites and increased DNA main stage Tm by 18 degrees C, and Tm of poly(dA)poly(dT) increased by 40 degrees C, in comparison with the same polymers without porphyrin. ZnTOEpyp(4) binds with DNA and poly(dA)poly(dT) in two modes--strong and weak ones. In the range of r from 0.005 to 0.08 both modes were fulfilled, and in the range of r from 0.165 to 0.25 only one mode--strong binding--took place. The weak binding is characterized with shifting of Tm by some grades, and for the strong binding Tm shifts by approximately 30-40 degrees C. Invariability of DeltaHm of DNA and poly(dA)poly(dT), and sharp increase of Tm in the range of r from 0.08 to 0.25 for thymus DNA and 0.01-0.2 for poly(dA)poly(dT) we interpret as entropic character of these complexes melting. It was suggested that this entropic character of melting is connected with forcing out of H2O molecules from AT sites by ZnTOEpyp(4) and with formation of outside stacking at the sites of binding. Four-fold decrease of calf thymus DNA melting range width DeltaTm caused by increase of added ZnTOEpyp(4) concentration is explained by rapprochement of AT and GC pairs thermal stability, and it is in agreement with a well-known dependence, according to which DeltaT approximately TGC-TAT for DNA obtained from higher organisms (L. V. Berestetskaya, M. D. Frank-Kamenetskii, and Yu. S. Lazurkin. Biopolymers 13, 193-205 (1974)). Poly (d(A-C))poly(d(G-T)) in the presence of ZnTOEpyp(4) gives only one mode of weak binding. The conclusion is that binding of ZnTOEpyp(4) with DNA depends on its nucleotide sequence.     

Purification and characterization of the DNA polymerase and RNase H activities in Moloney murine sarcoma-leukemia virus.

Two RNase H (RNA-DNA hybrid ribonucleotidohydrolase, EC 3.1.4.34) activities separable by Sephadex G-100 gel filtration were identified in lysates of Moloney murine sarcoma-leukemia virus (MSV). The larger enzyme, which we have called RNase H-I, represented about 10% of the RNase H activity in the virion. RNase H-I (i) copurified with RNA-directed DNA polymerase from the virus, (ii) had a sedimentation coefficient of 4.4S (corresponds to an apparent mol wt of 70,000), (iii) required Mn-2+ (2 mM optimum) for activity with a [3-h]poly(A)-poly(dT) substrate, (iv) eluted from phosphocellulose at 0.2 M KC1, and (v) degraded [3-H]poly(A)-poly(dT) and [3-H]poly(C)-poly(dG) at approximately equal rates. The smaller enzyme, designated RNase H-II, which represented the majority of the RNase H activity in the virus preparation, was shown to be different since it (i) had no detectable, associated DNA polymerase activity, (ii) had a sedmimentation coefficient of 2.6S (corresponds to an apparent mol wt of 30,000), (iii) preferred Mg-2+ (10 to 15 mM optimum) over Mn-2+ (5 to 10 mM optimum) 2.5-fold for the degradation of [3-H]poly(A)-poly(dT), and (iv) degraded [3-H]poly(A)-poly(dT) 6 and 60 times faster than [3-H]poly(C)-poly(dG) in the presence of Mn-2+ and Mg-2+, respectively. Moloney MSV DNA polymerase (RNase H-I), purified by Sephadex G-100 gel filtration followed by phosphocellulose, poly(A)-oligo(dT)-cellulose, and DEAE-cellulose chromatography, transcribed heteropolymeric regions of avian myeloblastosis virus 70S RNA at a rate comparable to avian myeloblastosis virus DNA polymerase purified by the same procedure.     

Comparison of base inclination in ribo-GC and deoxybribo-GC polymers,and synthesis of poly(rGrC)-poly(rGrC)

The inclination angle between the base normal and the helix axis, and the axes around which the bases incline, are measured for ribo-GC polymers in buffer by using flow linear dichroism (LD), and compared to measurements for deoxyribo-GC polymers in buffer and under dehydrating conditions. A new method is designed to synthesize poly(rGrC) -poly(rGrC), which is not available commercially, in large quantities. The LD of this RNA reveals inclination angles that are similar to the B-form DNA in buffer, although the axes are different. The CD of poly(dGdC)-poly(dGdC) under the dehydrating conditions is similar to poly(rGrC)-poly(rGrC), indicating it is in the A form, and the LD gives larger inclination angles than either the B form or the corresponding RNA. Poly(dG)-poly(dC) is in the A form in buffer. Comparison among poly(rG)-poly(rC) in buffer, and poly (dG)-poly(dC) in buffer and under dehydrating conditions, reveals similar inclination angles and axes, although the LD shows that the DNA has the largest inclination angles. Except for poly(rGrC)-poly(rGrC), which has a unique reduced dichroism, all the axes for G are similar, as are the axes for C. © 1995 John Wiley & Sons, Inc.     

Poly(dG)-poly(dC) DNA appears shorter than poly(dA)-poly(dT) and possibly adopts an A-related conformation on a mica surface under ambient conditions

Three types of DNA: approximately 2700 bp polydeoxyguanylic olydeoxycytidylic acid [poly(dG)-poly(dC)], approximately 2700 bp polydeoxyadenylic polydeoxythymidylic acid [poly(dA)-poly(dT)] and 2686 bp linear plasmid pUC19 were deposited on a mica surface and imaged by atomic force microscopy. Contour length measurements show that the average length of poly(dG)-poly(dC) is approximately 30% shorter than that of poly(dA)-poly(dT) and the plasmid. This led us to suggest that individual poly(dG)-poly(dC) molecules are immobilized on mica under ambient conditions in a form which is likely related to the A-form of DNA in contrast to poly(dA)-poly(dT) and random sequence DNA which are immobilized in a form that is related to the DNA B-form.     

Physical properties of inner histone-DNA complexes.

Chicken-erythrocyte inner histone tetramer has been complexed with several natural and synthetic DNA duplexes by salt-gradient dialysis at various protein/DNA ratios. The resulting complexes, in low-ionic-strength buffer, have been examined by electron microscopy, circular dichroism, and thermal denaturation. Electron microscopy reveals nucleosomes (nu bodies) randomly arranged along DNA fibers, including poly(dA-dT)-poly(dA-dT), poly(dI-dC)-poly(dI-dC), but not poly(dA)-poly(dT). Circular dichroism studies showed prominent histone alpha-helix and "suppression" of nucleic acid ellipticity (lambda less than 240 nm). Thermal denaturation experiments revealed Tm behavior comparable to that of H1- (or H5-) depleted chromatin. Tm III and Tm IV increased linearly with G + C%(natural DNAs), but were virtually independent of the histone/DNA ratio; therefore, the melting of nucleosomes along a DNA chain is insensitive to adjacent "spacer" DNA lengths. This suggests that Tm III and Tm IV arise from the melting of different domains of DNA associated with the core nu body.     

Poly(dG)-poly(dC) sequences, under torsional stress, induce an altered DNA conformation upon neighboring DNA sequences

Supercoiled plasmid DNAs (at bacterial superhelical density) harboring the homopurine-homopyrimidine sequence, poly(dG)-poly(dC), were reacted with bromoacetaldehyde (BAA), a reagent that reacts with unpaired DNA bases. Not only did the poly(dG)-poly(dC) sequence react with BAA but, surprisingly, neighboring sequences located 3' to the contiguous G sequences also reacted. The altered conformation in the poly(dG)-poly(dC) sequence and in the neighboring sequence occurred in the same supercoiled plasmid DNA molecule. Furthermore, the occurrence of an "unpaired" conformation in the neighboring sequence is strictly due to a positional effect, since it is observed when the poly(dG)-poly(dC) segment is adjacent to a variety of neighboring sequences.     

The interaction of ethidium with synthetic double-stranded polynucleotides at low ionic strength.

The interaction of ethidium with synthetic DNA and RNA double-stranded polymers at 0.01 M ionic strength, pH 7.0, has been studied by fluorimetry at low drug to nucleotide ratios. Binding constants have been calculated assuming an excluded-neighbouring site model for the interaction of ethidium with double-stranded polymers. The values obtained are poly d(AT).poly d(AT), 9.5 X 10(6) M-1; poly dA.poly dT, 6.5 X 10(5) M-1; poly d(GC).poly d(GC), 9.9 X 10(6) M-1; poly dG,poly dC, 4.5 X 1-(6) M-1; poly d(AC); poly d(GT), 9.8 X 10(6) M-1; poly d(AG).poly d(CT), 1.3 X 10(6) M-1; poly rA.poly rU, 4.1 X 10(7) M-1. The displacement of ethidium from poly d(AT).poly d(AT) by 9-aminoacridine and an acridine-containing antitumor agent (NSC 156303; 4'-(9-acridinylamino)methanesulphon-m-anisidide) has also been examined.     

Comparison of base inclination in ribo-AU and deoxyribo-AT polymers

The inclination angle between the base normal and the helix axis is measured for ribo-AU polymers by using flow linear dichroism (LD), and compared to measurements for deoxyribo-AT polymers under dehydrating conditions. The CD of the DNA polymers under the dehydrating conditions is not the same as the corresponding RNA polymers, which are presumed to be in the A form. However, the LD indicates that poly(dAdT)-poly(dAdT) can assume the A form in 80% 2,2,2-trifluoroethanol, although poly(dA)-poly(dT) retains B form structure in this dehydrating solvent. The inclination angles are similar for B form poly(dAdT)-poly(dAdT) and poly(dA)-poly(dT), and these parameters are also similar for A form poly(rArU) -poly(rArU) and poly(rA) -poly(rU). All the inclination axes are similar. © 1995 John Wiley & Sons, Inc.     

Fluorescent d(CGCGAATTCGCG): characterization of major groove polarity and study of minor groove interactions through a major groove semantophore conjugate.

The major and minor groove in duplex DNA are sites of specific molecular recognition by DNA-binding agents such as proteins, drugs and metal complexes and have functional significance. In view of this, understanding of the inherent differences in their environment and the allosteric information transfer between them induced by DNA-binding agents assumes importance. Site-specific incorporation of 5-aminodansyl-dU, (U*) in oligonucleotides d(CGCGAAU*TCGCG) and d(CGCGAATU*CGCG) leads to fluorogenic nucleic acids, in which the reporter group resides in the major groove. The fluorescent observables from such a probe are used to estimate the dielectric constant of the major groove to be approximately 55D, in comparison to the reported non polar environment of the minor groove (approximately 20D) in poly d[AT]-poly d[AT]. An exclusive minor groove event such as DNA-netropsin association can be quantitatively monitored by fluorescence of the dansyl moiety located in the major groove. This suggests existence of an information network among the two grooves. The fluorescent DNA probes as reported here may have potential applications in the study of structural polymorphisms in DNA, DNA-ligand interactions and triple helix structure.     

Nucleosomal structure of two Drosophila melanogaster simple satellites

Nucleosomes have been fractionated on nondenaturing polyacrylamide gels, and nucleosome subtypes containing the Drosophila melanogaster specific protein D1 and ubiquitinated core histone H2A were identified by solubility in 0.1 M NaCl before nucleoprotein gel electrophoresis. Nucleosomes which contain DNA complementary to the 1.672 density simple satellite (sequence-AATAT-) bind protein D1, as demonstrated by two-dimensional hybridization mapping. This hybridization pattern allows the identification of D1 dinucleosomes, which, like D1 mononucleosomes, are reduced in mobility on the first dimension (nucleoprotein) gel by the addition of D1, an AT sequence-specific DNA-binding protein. The 1.705 density simple satellite (sequence-AAGAG-) is also found in nucleosomes, in a radically different subset from those of the -AATAT- DNA sequence. -AAGAG- nucleosomes do not contain D1 protein, but appear to be enriched in ubiquitinated core histone H2A. One-dimensional hybridization patterns suggest that -AAGAG- nucleosomal DNA is rapidly trimmed to a shorter DNA length than either bulk or -AATAT- nucleosomes.     

Dissymmetric recognition of the helical sense of deoxyribonucleic acid and evidence for binding of reporter molecules from the minor groove of DNA

Through the utilization of optically active DNP-derivatives of l- and d-proline, evidence is presented which suggests that nucleic acids exist as right-handed helices in solution. The results of ultraviolet absorption, circular dichroism, proton magnetic resonance (pmr), T_m of the helix-coil transition, viscometric, and binding studies are consistent with the above interpretation. It is shown that several types of DNA (i.e., salmon sperm, calf thymus, Micrococcus luteus, poly d(A-T)-poly d(A-T) and poly d(I-C)-poly d(I-C)) exist in a right-handed helical structure in solution. In addition, evidence is presented which strongly indicates that the 2,4-dinitroaniline ring of DNP-proline is intercalated between base-pairs of DNA and the prolyl side chain situated in the minor groove. Moreover, it is shown that the more sterically hindered DNP-derivatives exhibit a higher selectivity for A-T binding sites.     

Stopped-flow kinetic studies of actinomycin binding to DNAs.

Stopped-flow kinetic studies of the association of actinomycins with narural and synthetic DNA duplexes are presented. The actinomycins examined were D (C1), D lactam (in which the pentapeptide rings are closed by lactam instead of lactone linkages), X2, XObeta, and actinomine. The DNAs used included claf-thymus DNA, PM2, DNA, and two synthetic d(A-T)-lide copolymers containing 2,6-diaminopurine (DAP) in place of adenine residues, poly[d(DAP-T)]-poly[d(DAP-T)] and poly[d(DAP-A-T]-poly[d(DAP-A-T)]. Apparent equilibrium constants indicate that the DAP-containing polynucleotides bind actinomycin strongly. Comples formation of actinomycins D, D lactam, X2 and XObeta with these DNAs can be deconvoluted into five rate processes. These steps do not necessarily proceed to completion. The rates of two of these steps display a firstorder dependence on DNA concentration. The large negative entropies of activation of these steps suggest a high degree of restriction to freedom of motion on the respective transition states. The rates of the remaining three steps are independent of DNA concentration. Kinetic parameters of actinimycin binding to DNAs are presented and suggestions are made about some of the molecular evente believed to be responsible for the appearance of the five rate processes. For example, for DNA, poly[d(DAP-A-T)], and poly[d(DAP-T)], the observed order of apparent second-order rate constants, normalized to the concentration of actinomycin binding sites, suggests that binding of the antibiotic occurs most rapidly at binding sites (G-C of d DAT-T) near d(A-T) base pairs, where weakening of the double-helical conformation requires the least energy. Results obtained from studies of actinomycin D binding to heat-denatured poly[d(DAP-A-T)] and of actinomine and actinomycin D lactam binding to DNA suggest that the slow rate processes are related to an actinomycyl-pentapeptide-induced unwinding of the sugar-phosphate backbone of DNA accompanying insertion of the cyclic peptides into DNA.     

Binding of Hoechst 33258 and its Derivatives to DNA

Abstract

In the present work, we employed UV-VIS spectroscopy, fluorescence methods, and circular dichroism spectroscopy (CD) to study the interaction of dye Hoechst 33258, Hoechst 33342, and their derivatives to poly[d(AT)]·poly[d(AT)], poly(dA)·poly(dT), and DNA dodecamer with the sequence 5′-CGTATATATACG-3′. We identified three types of complexes formed by Hoechst 33258, Hoechst 33342, and methylproamine with DNA, corresponding to the binding of each drug in monomer, dimer, and tetramer forms. In a dimer complex, two dye molecules are sandwiched in the same place of the minor DNA groove. Our data show that Hoechst 33258, Hoechst 33342, and methylproamine also form complexes of the third type that reflects binding of dye associates (probably tetramers) to DNA. Substitution of a hydrogen atom in the ortho position of the phenyl ring by a methyl group has a little effect on binding of monomers to DNA. However it reduces strength of binding of tetramers to DNA. In contrast, a Hoechst derivative containing the ortho-isopropyl group in the phenyl ring exhibits a low affinity to poly(dA)·poly(dT) and poly[d(AT)]·poly[d(AT)] and binds to DNA only in the monomer form. This can be attributed to a sterical hindrance caused by the ortho-isopropyl group for side-by-side accommodation of two dye molecules in the minor groove. Our experiments show that mode of binding of Hoechst 33258 derivatives and their affinity for DNA depend on substituents in the ortho position of the phenyl ring of the dye molecule. A statistical mechanical treatment of binding of Hoechst 33258 and its derivatives to a polynucleotide lattice is described and used for determination of binding parameters of Hoechst 33258 and its derivatives to poly[d(AT)]·poly[d(AT)] and poly(dA)·poly(dT).     

Structure of a pleiomeric form of poly d(AT):poly d(AT)

A chemically simple polynucleotide duplex, poly d(AT):poly d(AT), has been trapped in a fibrous form with a complex helical secondary structure with a large (7.4 nm) axial repeat 24 nucleotides long. The motif which is repeated by the symmetry elements is a hexanucleotide in which two residues (both TpA) have the less common gauche minus conformation at C3'-O3' and consequently distinctive phosphate orientations. This reinforces earlier conclusions that PypPu nucleotides tend to have different shapes from PupPy nucleotides and that DNA surfaces may signal what base sequences lie beneath them. The morphological differences between this pleiomeric DNA polymer and closely-related, but more symmetrical allomorphs are just as great as those observed in short DNA fragments in crystals.     

Antisera to poly(A)-poly(U)-poly(I) contain antibody subpopulations specific for different aspects of the triple helix.

Rabbit antibodies to the triple-helical polynucleotide poly(A)-poly(U)-poly(I) were fractionated into three major antibody populations, each recognizing a different conformational feature of the triple-helical immunogen. Two distinct populations were purified from precipitates made with poly(A)-poly(U)-poly(U) and poly(A)-poly(I)-poly(I). The former reacted with double-stranded poly(A)-poly(U) or poly(I)-poly(C), and similar populations could be purified with either double-stranded form. The second population recognized the poly(A)-poly(I) region of the triple helix, and the third required all three strands for reactivity. These immunochemical studies suggest that the poly(A) and poly(U) have the same orientation in the triple-helicical poly(A)-poly(U)-poly(I) as in the double-helical poly(A)-poly(U), in which they have Watson-Crick base pairing.