Interaction of nucleic acids. VI. Interaction of purine with nucleic acids

The interaction of purine with DNA, tRNA, poly A, poly C, and poly A. poly U complex was investigated. In the presence of purine, the nucleic acids in coil form (such as denatured DNA, poly A and poly C in neutral solutions, or tRNA) have lower optical rotations. In addition, hydrodynamic studies indicate that in purine solutions the denatured DNA has a higher viscosity and a decreased sedimentation coefficient. These findings indicate that through interaction with purine, the bases along the poly-nucleotide chain are unstacked and are separated farther from each other, resulting in increased assymmetry (and possibly volume) of the whole polymer. Thus, the de-naturation effect of purine reported previously can be explained by this preferential interaction of purine with the bases of nucleic acids in coil form through a hydrophobic-costacking mechanism. Results from studies on optical rotation and helix-coil transition show that the interaction of purine is greater with poly A than with poly C. The influence of temperature, Mg⁺⁺ concentration, ionic strength, and purine concentration on the effect of purine on nucleic acid conformation has also been investigated. In all these situations the unraveling of nucleic acid conformation occurs at much lower temperatures (20–40°C lower) in the presence of purine (0.2–0.6M).     

The in vitro DNA-binding properties of purified nuclear lamin proteins and vimentin

The ability of purified nuclear lamin A, lamin B, lamin C, and vimentin from Ehrlich ascites tumor cells to bind nucleic acids was investigated in vitro via a quantitative filter binding assay. At low ionic strength, vimentin bound more nucleic acid than the nuclear lamins and showed a preference for G-containing nucleic acids. Nuclear lamins A and C were quite similar in their binding properties and bound G- and C-containing nucleic acids preferentially. The binding of poly(dT) by the lamins A and C was reduced in competition experiments by both poly(dG) and poly(dC), but not by poly(dA). Lamin B bound only oligo and poly(dG); no other nucleic acids tested were bound or could compete with the binding of oligo(dG). Vimentin, lamin A, and lamin C specifically bound a synthetic oligonucleotide human (vertebrate) telomere model. The Ka for vimentin (2.7 X 10(7) M-1) was approximately 10-fold higher than those for lamin A (2.8 X 10(6) M-1) and lamin C (2.9 X 10(6) M-1). Lamin B did not bind detectable amounts of the telomere model. Washing of lamin A- and lamin C-nucleic acid complexes, formed at low ionic strength, with solutions containing 150 mM KCl resulted in the elution of 30% of bound poly(dG)12-18 and 70% of bound synthetic oligonucleotide telomere model. These results, using purified individual proteins, are in good agreement with data from competition experiments with vimentin but are at odds with data obtained previously using a crude preparation of nuclear matrix proteins containing all three nuclear lamin proteins (Comings, D. E., and Wallack, A. S. (1978) J. Cell Sci. 34, 233-246). The nuclear lamins A and C and vimentin possess nucleic acid-binding properties that might permit their binding to specific base sequences and/or unique DNA structure, such as that observed for the binding of the telomere model. The significance of the higher affinity binding of nucleic acids by the cytoplasmic protein vimentin (compared with the nuclear lamins) remains to be elucidated.     

Shuffling of amino acid sequence: an important control in synthetic peptide studies of nucleic acid-binding domains. Binding properties of fragments of a conserved eukaryotic RNA binding motif.

We have used synthetic peptides to study a conserved RNA binding motif in yeast poly(A)-binding protein. Two peptides, 45 and 44 amino acids in length, corresponding to amino and carboxyl halves of a 90-amino acid RNA-binding domain in the protein were synthesized. While the amino-terminal peptide had no significant affinity for nucleic acids, the carboxyl-terminal peptide-bound nucleic acids with similar characteristics to that for the entire 577 residue yeast poly(A)-binding protein. In 100 mM NaCl, the latter peptide retained over 50% of the intrinsic binding free energy of the protein, as well as, similar RNA versus DNA binding specificity. However, shuffling of the sequence of this 44 residue peptide had surprisingly little effect on its nucleic acid binding properties suggesting the overriding importance of amino acid composition as opposed to primary sequence. Deletion studies on the 44 residue peptide with the "correct" sequence succeeded in identifying amino acids important for conferring RNA specificity and for increasing our understanding of the molecular basis for nucleic acid binding by synthetic peptides. The shuffled peptide study, however, clearly indicates that considerable caution must be exercised before extrapolating results of structure/function studies on synthetic peptide analogues to the parent protein.     

Biphasic helix–coil transition of the ethidium bromide·poly(dA-dT) and the propidium diiodide·poly(dA-dT) Complexes. Stabilization of base-pair regions centered about the intercalation site

The nonexchangeable base and sugar proton nmr resonances and the 260 and 278-nm uv-absorbance bands of the nucleic acid were utilized to monitor the temperature-dependent duplex-to-strand transition of the alternating purine–pyrimidine deoxyribopolynucleotide poly(dA-dT) in the absence and presence of ethidium bromide (EB) at phosphate/drug = 50, 28, and 15 and propidium diiodide (PI) at P/D = 50, 25, 15, 10, and 5 in 0.1 M salt between 50° and 100°C. The nmr and optical methods monitor a biphasic duplex-to strand transition for the drug–poly(dA-dT) complexes. We have monitored the dissociation of the drug from the complex at the ethidium bromide phenanthridine ring and side-chain proton nmr resonances and the propidium diiodide 494 and 535-nm uv-absorbance bands and demonstrate that dissociation of the drug corresponds to the higher temperature transition in the biphasic nucleic acid melting curves. The lower temperature cooperative transition is assigned to the opening of drug-free AT base-pair regions in the drug–poly(dA-dT) complex and exhibits an increase in transition midpoint and a decrease in cooperativity with increasing drug concentration. The higher temperature cooperative transition is assigned to the opening of AT base-pair regions centered about the bound drug in the complex and exhibits an increase in the transition midpoint on raising the drug concentration. The large upfield shifts of the phenanthridine ring (but not side chain) protons of ethidium bromide on complex formation demonstrate intercalation of the drug between base pairs of the poly(dA-dT) duplex. The nucleic acid base and sugar resonances of poly(dA-dT) in 0.1 M phosphate undergo chemical shift changes between 0° and 50°C indicative of premelting conformational transition(s).     

Binding of Nucleic Acids to Intermediate Filaments of the Vimentin Type and Their Effects on Filament Formation and Stability

Abstract

Guanine-rich polynucleotides such as poly(dG), oligo(dG)_12–18 or poly(rG) were shown to exert a strong inhibitory effect on vimentin filament assembly and also to cause disintegration of preformed filaments in vitro. Gold-labeled oligo(dG)₂₅ was preferentially localized at the physical ends of the aggregation and disaggregation products and at sites along filaments with a basic periodicity of 22.7 nm. Similar effects were observed with heat-denatured eukaryotic nuclear DNA or total rRNA although these nucleic acids could affect filament formation and structure only at ionic strengths lower than physiological. However, whenever filaments were formed or stayed intact, they appeared associated with the nucleic acids. These electron microscopic observations were corroborated by sucrose gradient analysis of complexes obtained from preformed vimentin filaments and radioactively labeled heteroduplexes. Among the duplexes of the DNA type, particularly poly(dG)·poly(dC), and, of those of the RNA type, preferentially poly(rA)·poly(rU), were carried by the filaments with high efficiency into the pellet fraction. Single-stranded 18S and 28S rRNA interacted only weakly with vimentin filaments. Nevertheless, in a mechanically undisturbed environment, vimentin filaments could be densely decorated with intact 40S and 60S ribosomal subunits as revealed by electron microscopy. These results indicate that, in contrast to single-stranded nucleic acids with their compact random coil configuration, double-stranded nucleic acids with their elongated and flexible shape have the capability to stably interact with the helically arranged, surface-exposed amino-terminal polypeptide chains of vimentin filaments. Such interactions might be of physiological relevance in regard to the transport and positioning of nucleic acids and nucleoprotein particles in the various compartments of eukaryotic cells. Conversely, nucleic acids might be capable of affecting the cytoplasmic organization of vimentin filament networks through their filament-destabilizing potentials.     

Binding of eucaryotic elongation factor Tu to nucleic acids

The binding of eucaryotic elongation factor Tu (eEF-Tu) to nucleic acids was investigated. eEF-Tu binds to a variety of different nucleic acids with high affinity, showing a strong preference for 18 S and 28 S rRNA over transfer RNA and for ribose-containing polymers over polydeoxyribonucleotides. The factor binds at multiple sites on 28 S rRNA without strong cooperativity. eEF-Tu binds strongly to poly(G) and poly(U) but weakly, if at all, to poly(A) and poly(C). Experiments employing an airfuge demonstrate that eEF-Tu can form a quaternary complex containing the factor, 28 S rRNA, aminoacyl-tRNA, and GTP. The existence of two distinct RNA binding sites on eEF-Tu suggests that rRNA may play a role in the recognition of eEF-Tu.aminoacyl-tRNA.GTP complexes by polysomes. Support for this suggestion comes from experiments which show that poly(G) inhibits the factor-dependent binding of aminoacyl-tRNA to mRNA-programmed 80 S ribosomes. In addition, it is shown that eEF-Tu possesses an intrinsic GTPase activity which is stimulated significantly by 28 S rRNA, poly(G), and poly(U). The binding of eEF-Tu to poly(G) lowers the activation energy for eEF-Tu GTPase from 74.3 to 65.9 kJ . mol-1 and approximately doubles the Vmax of the enzymatic reaction. The results are discussed in relation to the binding of eEF-Tu to ribosomes during protein synthesis.     

Inhibition of some DNA polymerase activities from cultured Burkitt cells by thiolated ribonucleic acids

Although unmodified poly C and unmodified ribosomal RNA showed little ( < 20%) or no inhibition of 6–7S cytoplasmic, 3–4S cytoplasmic and 3–4S chromatin-associated DNA-directed DNA polymerases and of RNase sensitive endogenous DNA polymerase and DNA-directed DNA polymerase activity associated with a particulate material (p = 1.16 ? 1.18 g/ml) from Burkitt cells the thiolated poly C and thiolated RNA were strongly inhibitory (70–97%). Moreover, the thiolated nucleic acids were more inhibitory to 6–7S enzyme than to 3–4S enzyme. Thiolation of nucleic acids thus appears to be a potentially important procedure for the development of agents which may be selective against certain polymerases.     

Influence of cyanine dye structure on self-aggregation and interaction with nucleic acids: a kinetic approach to TO and BO binding

Kinetics and equilibria of cyanine dyes thiazole orange (TO) and benzothiazole orange (BO) self-aggregation and binding to CT-DNA are investigated in aqueous solution at 25 degrees C and pH 7. Absorbance spectra and T-jump experiments reveal that BO forms J-aggregates while TO forms more stable H-aggregates. Fluorescence and absorbance titrations show that TO binds to DNA more tightly than BO. TO stacks externally to DNA for low polymer-to-dye concentration ratios (C(P)/C(D)) while dye intercalation occurs for high values of C(P)/C(D). T-jump and stopped-flow experiments performed at high C(P)/C(D) agree with reaction scheme D+S <=> D,S <=> DS(I) <=> DS(II) where the precursor complex D,S evolves to a partially intercalated complex DS(I) which converts to the more stable intercalate DS(II). Non-electrostatic forces play a major role in D,S stabilization. Last step is similar for both dyes suggesting accommodation of the common benzothiazole residue between base pairs. Experiments using poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC) confirm base pair preference for TO.     

Rheological and structural characterization of the interactions between cyclodextrin compounds and hydrophobically modified alginate

Interactions in semidilute solutions of a hydrophobically modified alginate (HM-alginate) in the presence of hydroxypropyl-beta-cyclodextrin (HP-beta-CD) monomer or a beta-cyclodextrin polymer (poly(beta-CD)) have been characterized at different temperatures with the aid of rheology and small-angle neutron scattering (SANS). The viscosity results for the HM-alginate/HP-beta-CD system reveal progressive deactivation of the hydrophobic associations as the concentration of HP-beta-CD increases. For the HM-alginate/poly(beta-CD) system, on the other hand, addition of poly(beta-CD) sets up bridges between adjacent polymer chains and thereby strengthens the associative network. A novel shear-thickening effect is observed at fairly high shear rates for the HM-alginate/poly(beta-CD) system, and this feature is influenced by temperature. Elevated temperature induces higher chain mobility and the formation of weaker network associations. Analyses of the SANS data disclosed that the association strength in HM-alginate/poly(beta-CD) mixtures increases strongly with increasing cosolute concentration, whereas no effect or a moderate weakening of the strength can be traced in HM-alginate/HP-beta-CD solutions upon addition of HP-beta-CD. The value of the correlation length xi is virtually not affected by the addition of cosolute for the HM-alginate/poly(beta-CD) system, whereas the decoupling of hydrophobic moieties of the polymer upon addition of HP-beta-CD gives rise to a smaller value of xi, suggesting that the size of the heterogeneity patches is reduced. The SANS results suggest that compact association structures are formed in the HM-alginate/poly(beta-CD) solutions.     

Specific binding and self-structure induction to poly(A) by the cytotoxic plant alkaloid sanguinarine

The cytotoxic plant alkaloid sanguinarine was found to bind preferentially and strongly to single stranded poly(A) with an association constant (K(a)) in the range 3.6-4.6 x 10(6) M(-1) in comparison to several nucleic acids. The binding induced unique self-structure formation in poly(A) that showed cooperative melting transition in circular dichroism, absorbance, and differential scanning calorimetry studies. The alkaloid binding was characterized to be intercalation as revealed from fluorescence quenching experiments and was predominantly enthalpy driven as revealed from isothermal titration calorimetry. Sanguinarine is the first and only natural product so far known to induce a self-structure formation in poly(A).     

Tyrosine mutant helps define overlapping CD bands from fd gene 5 protein · nucleic acid complexes

We used a mutant gene 5 protein (g5p) to assign and interpret overlapping CD bands of protein · nucleic acid complexes. The analysis of overlapping protein and nucleic acid CD bands is a common challenge for CD spectroscopists, since both components of the complex may change upon binding. We have now been able to more confidently resolve the bands of nucleic acids complexed with the fd gene 5 protein by exploiting a mutant gene 5 protein that has an insignificant change in tyrosine optical activity at 229 nm upon binding to nucleic acids. We have studied the interactions of the mutant Y34F g5p (Tyr-34 substituted with phenylalanine) with poly[r(A)], poly[d(A)], and fd single-stranded DNA (ssDNA). Our results showed the following: (1) The 205–300 nm spectrum of poly[r(A)] saturated with the Y34F mutant (P/N = 0.25) was essentially the sum of the spectra of poly[r(A)] at a high temperature plus the spectrum of the free protein, except for a minor negative band at 257 nm. (2) The spectra of poly[d(A)] and fd ssDNA saturated with the mutant protein at a P/N = 0.25, minus the spectra of the free nucleic acids at a high temperature, also essentially equaled the spectrum of the free protein in the 205–245 nm region. (3) While the overall secondary structure of the Y34F protein did not change upon binding to any of these nucleic acids, there could be changes in the environment of individual aromatic residues. (4) Nucleic acids complexed with the g5p are unstacked (as if heated) and (in the cases of the DNAs) perturbed as if part of a dehydrated double-stranded DNA. (5) Difference spectra revealed regions of the spectrum specific for the particular nucleic acid, the protein, and whether g5p was bound to DNA or RNA. © 1997 John Wiley and Sons, Inc. Biopoly 42: 337–348, 1997     

Separation of nucleic acids and chromatin proteins by hydrophobic interaction chromatography

A procedure by which chromatin proteins (histones and non-histones) can be rapidly separated from nucleic acids by hydrophobic interaction chromatography is described. The procedure is carried out under non-rigorous conditions that must be assumed to induce little irreversible change in the biological properties of most proteins. More than 90% (w/w) of the chromatin proteins can be retained by hydrophobic interaction while nucleic acids pass quantitatively through the columns. By gradient elution of the columns the histones can be divided into fractions containing H1, H2A/H2B and H3/H4, and at the same time a subfractionation of the non-histone proteins is obtained. Protein recovery depends on the type of column used, but exceeds 80% (w/w) with even the most strongly binding hydrophobic matrix investigated.     

The binding of polynucleotides to the DNA polymerase of avian myeloblastosis virus.

The interaction between avian myeloblastosis virus DNA polymerase and synthetic nucleic acids was studied by an adaptation of the membrane filter binding technique. Bacillus subtilis DNA was used as a substrate for the binding reaction and was retained on the filters in the presence of the viral polymerase. The polymerase activity was demonstrated to be retained on the filter in either the presence or absence of the bacterial DNA. Characterization of the polymerase-DNA interaction demonstrated a marked similarity to previous data regarding the binding of Escherichia coli DNA-dependent RNA polymerase to nucleic acids when studied using related techniques. In contrast, the association between methylated bovine serum albumin and the B. subtilis DNA was found to differ significantly in both reaction stoichiometry and stability. Synthetic polynucleotides were shown to inhibit the binding of the bacterial DNA to the viral DNA polymerase and poly 2′-fluoro-2′-deoxyuridylic acid was found to be the most potent inhibitor of this reaction. Results from the binding-inhibition studies correlated well with studies concerning the inhibition of enzyme activity and it is concluded that the inhibitory polynucleotides act by interfering with binding of nucleic acid template to the viral enzyme.     

Single-stage chromatographic clarification of Chinese Hamster Ovary cell harvest reduces cost of protein production

A single-stage clarification was developed using a single-use chromatographic clarification device (CCD) to recover a recombinant protein from Chinese Hamster Ovary (CHO) harvest cell culture fluid (HCCF). Clarification of a CHO HCCF is a complex and costly process, involving multiple stages of centrifugation and/or depth filtration to remove cells and debris and to reduce process-related impurities such as host cell protein (HCP), nucleic acids, and lipids. When using depth filtration, the filter train consists of multiple filters of varying ratios, layers, pore sizes, and adsorptive properties. The depth filters, in combination with a 0.2-micron membrane filter, clarify the HCCF based on size-exclusion, adsorptive, and charge-based mechanisms, and provide robust bioburden control. Each stage of the clarification process requires time, labor, and utilities, with product loss at each step. Here, use of the 3M™ Harvest RC Chromatographic Clarifier, a single-stage CCD, is identified as an alternative strategy to a three-stage filtration train. The CCD results in less overall filter area, less volume for flushing, and higher yield. Using bioprocess cost modeling, the single-stage clarification process was compared to a three-stage filtration process. By compressing the CHO HCCF clarification to a single chromatographic stage, the overall cost of the clarification process was reduced by 17%–30%, depending on bioreactor scale. The main drivers for the cost reduction were reduced total filtration area, labor, time, and utilities. The benefits of the single-stage harvest process extended throughout the downstream process, resulting in a 25% relative increase in cumulative yield with comparable impurity clearance.     

The hydration of nucleic acid duplexes as assessed by a combination of volumetric and structural techniques

Using high precision densimetric and ultrasonic measurements, we have determined, at 25°C, the apparent molar volumes ΦV and the apparent molar compressibilities ΦK_S of four nucleic acid duplexes—namely, the DNA duplex, poly(dIdC)poly(dIdC); the RNA duplex, poly(rA)poly(rU); and the two DNA/RNA hybrid duplexes, poly(rA)poly(dT) and poly(dA)poly(rU). Using available fiber diffraction data on these duplexes, we have calculated the molecular volumes as well as the solvent‐accessible surface areas of the constituent charged, polar, and nonpolar atomic groups. We found that the hydration properties of these nucleic acid duplexes do not correlate with the extent and the chemical nature of the solvent‐exposed surfaces, thereby suggesting a more specific set of duplex–water interactions beyond general solvation effects. A comparative analysis of our volumetric data on the four duplexes, in conjunction with available structural information, suggests the following features of duplex hydration: (a) The four duplexes exhibit different degrees of hydration, in the order poly(dIdC)poly(dIdC) > poly(dGdC)poly(dGdC) > poly(dAdT)poly(dAdT) ≈ poly(dA)poly(dT). (b) Repetitive AT and IC sequences within a duplex are solvated beyond general effects by a spine of hydration in the minor groove, with this sequence‐specific water network involving about 8 additional water molecules from the second and, perhaps, even the third hydration layers. (c) Repetitive GC and IC sequences within a duplex are solvated beyond general effects by a “patch of hydration” in the major groove, with this water network involving about 13 additional water molecules from the second and, perhaps, even the third hydration layers. (d) Random sequence, polymeric DNA duplexes, which statistically lack extended regions of repetitive AT, GC, or IC sequences, do not experience such specific enhancements of hydration. Consequently, consistent with our previous observations (T. V. Chalikian, A. P. Sarvazyan, G. E. Plum, and K. J. Breslauer, Biochemistry, 1994, Vol. 33, pp. 2394–2401), duplexes with approximately 50% AT content exhibit the weakest hydration, while an increase or decrease from this AT content causes enhancement of hydration, either due to stronger hydration of the minor groove (an increase in AT content) or due to stronger hydration of the major groove (an increase in GC content). (e) In dilute aqueous solutions, a B‐DNA duplex is more hydrated than an A‐DNA duplex, a volumetric‐based conclusion that is in agreement with previous results obtained on crystals, fibers, and DNA solutions in organic solvent–water mixtures. (f) the A‐like, RNA duplex poly(rA)poly(rU) and the structurally similar A‐like, hybrid duplex poly(rA)poly(dT), exhibit similar hydration properties, while the structurally distinct A‐like, hybrid duplex poly(rA)poly(dT) and non‐A‐like, hybrid duplex poly(dA)poly(rU) exhibit differential hydration properties, consistent with structural features dictating hydration characteristics. We discuss how volumetric characterizations, in conjunction with structural studies, can be used to describe, define, and resolve the general and sequence/conformation‐specific hydration properties of nucleic acid duplexes. © 1999 John Wiley & Sons, Inc. Biopoly 50: 459–471, 1999     

Thermodynamics of nucleic acid "shape readout" by an aminosugar

Recognition of nucleic acids is important for our understanding of nucleic acid structure as well as for our understanding of nucleic acid-protein interactions. In addition to the direct readout mechanisms of nucleic acids such as H-bonding, shape recognition of nucleic acids is being increasingly recognized as playing an equally important role in DNA recognition. Competition dialysis, UV, flourescent intercalator displacement (FID), computational docking, and calorimetry studies were conducted to study the interaction of neomycin with a variety of nucleic acid conformations (shapes). At pH 5.5, the results suggest the following. (1) Neomycin binds three RNA structures [16S A site rRNA, poly(rA)·poly(rA), and poly(rA)·poly(rU)] with high affinities (K(a) ~ 10(7) M(-1)). (2) The binding of neomycin to A-form GC-rich oligomer d(A(2)G(15)C(15)T(2))(2) has an affinity comparable to those of RNA structures. (3) The binding of neomycin to DNA·RNA hybrids shows a 3-fold variance that can be attributed to their structural differences [for poly(dA)·poly(rU), K(a) = 9.4 × 10(6) M(-1), and for poly(rA)·poly(dT), K(a) = 3.1 × 10(6) M(-1)]. (4) The interaction of neomycin with DNA triplex poly(dA)·2poly(dT) yields a binding affinity (K(a)) of 2.4 × 10(5) M(-1). (5) Poly(dA-dT)(2) shows the lowest association constant for all nucleic acids studied (K(a) < 10(5)). (6) Neomycin binds to G-quadruplexes with K(a) values of ~10(4)-10(5) M(-1). (7) Computational studies show that the decrease in major groove width in the B to A transition correlates with increasing neomycin affinity. Neomycin's affinity for various nucleic acid structures can be ranked as follows: RNAs and GC-rich d(A(2)G(15)C(15)T(2))(2) structures > poly(dA)·poly(rU) > poly(rA)·poly(dT) > T·A-T triplex, G-quadruplex, B-form AT-rich, or GC-rich DNA sequences. The results illustrate the first example of a small molecule-based "shape readout" of different nucleic acid conformations.     

Isolation and characterization of poly(A)-containing polyoma "early" and "late" messenger RNAs.

During late lytic infection of mouse kidney cell cultures polyoma 16S and 19S (late 19S RNA) were isolated by oligo(dT)-cellulose chromatography. Approximately 60-80% of total cytoplasmic polyoma RNA contained tracts of poly(A) which were retained by oligo(dT)-cellulose. Early in lytic infection when viral DNA synthesis and the production of capsid protein are blocked by the addition of 5-fluorodeoxyuridine, approximately 100% of polyoma "early" 19S RNA was quantitatively retained by oligo(dT)-cellulose indicating the presence of poly(A) tracts on most 19S mRNA molecules. In addition, 2 classes polyoma RNA, synthesized after the onset of cellular RNA synthesis under conditions where DNA synthesis is inhibited with 5-fluorodeoxyuridine, were found to contain tracts of poly(A). These species sedimenting at 16S and 19S in aqueous sucrose density gradients were also quantitatively retained by oligo (dT)-cellulose.     

Binding properties of avian myeloblastosis virus DNA polymerases to nucleic acid affinity columns.

A new method for the analysis and purification of the RNA-directed DNA polymerase of RNA tumor viruses has been developed. This nucleic acid affinity chromatography system utilizes an immobilized oligo (dT) moiety annealed with poly (A). The alpha and alphabeta DNA polymerases of avain myeloblastosis virus bound effectively to poly (A) oligo (dT)-cellulose. Alpha DNA polymerase did not bind effectively to poly (A) oligo (dT)-cellulose, poly (A)-cellulose, or to cellulose. Alphabeta bound to oligo (dT)-cellulose and cellulose at the same extent (approximately 30%), indicating that this enzyme did not bind specifically to the oligo (DT) moiety only. However, alphabeta bound to poly (A)-cellulose two to three times better than to cellulose itself, showing that alphabeta could bind to poly (A) without a primer. Alphabeta DNA polymerase also bound to poly (C)-cellulose, whereas alpha did not. These data show that the alpha DNA polymerase is defective in binding to nucleic acids if the beta subunit is not present. Data is presented which demonstrates that the alphabeta DNA polymerase bound tighter to poly (A). oligo (DT)-cellulose and to calf thymus DNA-cellulose than the alpha DNA polymerase, suggesting that the beta subunit or, at least part of it is responsible for this tighter binding. In addition, alphabeta DNA polymerase is able to reversibly transcribe avian myeloblastosis virus 70S RNA approximately fivefold faster than alpha DNA polymerase in the presence of Mg2+ and equally efficient in the presence of Mn2+. alpha DNA polymerase transcribed 9S globin m RNA slightly better than alphabeta with either metal ion.     

Catalytic convergence of manganese and iron lipoxygenases by replacement of a single amino Acid

Lipoxygenases (LOXs) contain a hydrophobic substrate channel with the conserved Gly/Ala determinant of regio- and stereospecificity and a conserved Leu residue near the catalytic non-heme iron. Our goal was to study the importance of this region (Gly(332), Leu(336), and Phe(337)) of a lipoxygenase with catalytic manganese (13R-MnLOX). Recombinant 13R-MnLOX oxidizes 18:2n-6 and 18:3n-3 to 13R-, 11(S or R)-, and 9S-hydroperoxy metabolites (～80-85, 15-20, and 2-3%, respectively) by suprafacial hydrogen abstraction and oxygenation. Replacement of Phe(337) with Ile changed the stereochemistry of the 13-hydroperoxy metabolites of 18:2n-6 and 18:3n-3 (from ～100% R to 69-74% S) with little effect on regiospecificity. The abstraction of the pro-S hydrogen of 18:2n-6 was retained, suggesting antarafacial hydrogen abstraction and oxygenation. Replacement of Leu(336) with smaller hydrophobic residues (Val, Ala, and Gly) shifted the oxygenation from C-13 toward C-9 with formation of 9S- and 9R-hydroperoxy metabolites of 18:2n-6 and 18:3n-3. Replacement of Gly(332) and Leu(336) with larger hydrophobic residues (G332A and L336F) selectively augmented dehydration of 13R-hydroperoxyoctadeca-9Z,11E,15Z-trienoic acid and increased the oxidation at C-13 of 18:1n-6. We conclude that hydrophobic replacements of Leu(336) can modify the hydroperoxide configurations at C-9 with little effect on the R configuration at C-13 of the 18:2n-6 and 18:3n-3 metabolites. Replacement of Phe(337) with Ile changed the stereospecific oxidation of 18:2n-6 and 18:3n-3 with formation of 13S-hydroperoxides by hydrogen abstraction and oxygenation in analogy with soybean LOX-1.