Changes in density of DNA after interaction with dipicolinic acid and its possible role in spore heat resistance

We examined the effect of interacting dipicolinic acid and its calcium chelate on the wet and dry density of DNA. Complexes are produced whose densities are different from those of the individual components. Also, we observed two modes of binding, one strong the other weak, between DPA or CaDPA and DNA. The strength of the binding modes was reflected in the rate of dissolution of the complexes as monitored by changes in wet density with time and temperature. We conclude from these and other data in the literature that the interaction of dipicolinic acid with DNA not only influences the spore wet density and the ratio of core/core⁺ cortex volume, but may also influence the spore heat resistance.     

The interaction of chromium with nucleic acids

Native and denatured calf thymus DNA, and homopolyribonucleotides were compared with respect to chromium and protein binding after an in vitro incubation with rat liver microsomes, NADPH, and chromium(VI) or chromium(III). A significant amount of chromium bound to DNA when chromium(VI) was incubated with the native or the denatured form of DNA in the presence of microsomes and NADPH. For both native and denatured DNA the amount of protein bound to DNA increased with the amount of chromium bound to DNA. Denatured DNA had much higher amounts of chromium and protein bound than native DNA. There was no interaction between chromium(VI) and either form of DNA in the absence of the complete microsomal reducing system. The binding of chrornium(III) to native or denatured DNA was small and relatively unaffected by the presence of microsomes and NADPH. The binding of chromium and protein to polyriboadenylic acid (poly(A)), polyribocytidylic acid (poly(C), polyri-boguanylic acid (poly(G)) and polyribouridylic acid (poly(U)) was determined after incubation with chromium(VI) in the presence of microsomes and NADPH. The magnitude of chromium and protein binding to the ribo-polymers was found to be poly(G) ? poly(A) ? poly(C) ? poly(U). These results suggest that the metabolism of chromium(VI) is necessary in order for chromium to interact significantly with nucleic acids. The metabolically-produced chromium preferentially binds to the base guanine and results in DNA-protein cross-links. These findings are discussed with respect to the proposed scheme for the carcinogenicity of chromium(VI). Keywords: DNA-protein cross-links — Chromium-guanine interaction-Microsomal reduction of chromate     

An affinity scale for the interaction of amino acid residues with nucleic acids

The affinity of amino acid residues to nucleic acids is probed by measurements of melting temperatures t_m for the helix–coil transition at various concentrations of amino acid amides. The increase of t_m on addition of ligand is described by the equation t_m = t*_m + αlog(1+K_tc_λ), where t*_m is the melting temperature in the absence of ligand, c_λ the ligand concentration, and K_t the “t_m-onset” constant, which is analogous to an equilibrium constant. It is shown that K_t is closely related to the affinity of the ligands to the double helix, whereas the slope α mainly reflects the preference of the ligand binding to the helix versus the coil form. In the case of the amino acid amides, α is found to be virtually independent of the nature of the side chain with few exceptions, e. g., aromatic amides. The t_m-onset constant, however, strongly depends on the nature of the amino acid side chain. For simple aliphatic amino acids, the relative free energy of binding decreases with increasing hydrophobic free energy, e.g., a high affinity is found for Gly-amide and a low affinity for Leu-amide. This relation is modified by functional groups like OH in Ser-amide. The helices poly[d(A-T)], ploy[d(I-C)]. and poly[d(A-C)]·poly[d(G-T)] exhibit similar affinity scales with relatively small variations. Our results demonstrate that the hydrophilic character of double helices at their surface disfavors binding of hydrophobic ligands unless special contacts can be formed. From our results we establish an affinity scale for the binding of amino acids to double helices.     

Determination of dipicolinic acid in bacterial spores by derivative spectroscopy

Dipicolinic acid was extracted from approximately 0.1 mg spores or 0.5 ml of sporulating culture with 20 mM HCl for 10 min at 100 degrees C. The suspension was diluted with 5 mM Ca2+, 100 mM Tris, pH 7.6, centrifuged, and the first derivative of the uv absorbance spectrum recorded from 275 nm to 285 nm. DPA concentration was determined from the difference between the maximum at 276.6 nm and the minimum at 280 nm. The use of the difference between two first derivative values removed possible interference from sloping baselines. Turbidity, nucleic acids, and bacteriological media did not interfere. Analysis time for four extracts was 4 min using a spectrophotometer reading at 0.1-nm intervals. Dipicolinate at 0.1 mM gave 0.184 absorbance/nm at 25 degrees C. The coefficient of variation was 1.5%, and the detection limit 1 microM.     

Models of interaction between nucleic acids and proteins. Hydrogen bonding of arginine with nucleic acid bases, phosphate groups and carboxylic acids.

Complex formation between the side chain of arginine and nucleic acid bases has been investigated by proton magnetic resonance in dimethylsulfoxide. Simultaneous formation of two hydrogen bonds leads to a selectivity of arginine interaction towards cytosine and guanine. A comparison is made of the interaction of arginine side chain with nucleic acid bases, phosphate and carboxylate anions. It is shown that interaction between carboxylate and arginine is stronger than between phosphate and arginine. These results are discussed with respect to the selective recognition of nucleic acid bases by arginine side chains and by the arginyl-glutamyl ion pair which could form in proteins interacting with nucleic acids.     

Dynamic properties of interaction between nucleic acid bases and models of amino acids

We have discussed the possible hydrogen bonding mode of adenine-uracil base dimers and peptide side chains, together with their characteristic effects on the stability of adenine-uracil base pairing in chloroform solution by using proton nuclear magnetic resonance methods, where acetamide and butyric acid were used as a model of residues of glutamine and glutamic acid, respectively, and were added to an adenine-uracil equimolar mixture. The stability of base pairing was affected significantly when the model compounds approached the adenine-uracil base pairs; that is, the Hoogsteen type was destroyed, while the Watson-Crick type was formed more favorably.     

Assessment of heat resistance of bacterial spores from food product isolates by fluorescence monitoring of dipicolinic acid release

This study is aimed at the development and application of a convenient and rapid optical assay to monitor the wet-heat resistance of bacterial endospores occurring in food samples. We tested the feasibility of measuring the release of the abundant spore component dipicolinic acid (DPA) as a probe for heat inactivation. Spores were isolated from the laboratory type strain Bacillus subtilis 168 and from two food product isolates, Bacillus subtilis A163 and Bacillus sporothermodurans IC4. Spores from the lab strain appeared much less heat resistant than those from the two food product isolates. The decimal reduction times (D values) for spores from strains 168, A163, and IC4 recovered on Trypticase soy agar were 1.4, 0.7, and 0.3 min at 105 degrees C, 120 degrees C, and 131 degrees C, respectively. The estimated Z values were 6.3 degrees C, 6.1 degrees C, and 9.7 degrees C, respectively. The extent of DPA release from the three spore crops was monitored as a function of incubation time and temperature. DPA concentrations were determined by measuring the emission at 545 nm of the fluorescent terbium-DPA complex in a microtiter plate fluorometer. We defined spore heat resistance as the critical DPA release temperature (Tc), the temperature at which half the DPA content has been released within a fixed incubation time. We found Tc values for spores from Bacillus strains 168, A163, and IC4 of 108 degrees C, 121 degrees C, and 131 degrees C, respectively. On the basis of these observations, we developed a quantitative model that describes the time and temperature dependence of the experimentally determined extent of DPA release and spore inactivation. The model predicts a DPA release rate profile for each inactivated spore. In addition, it uncovers remarkable differences in the values for the temperature dependence parameters for the rate of spore inactivation, DPA release duration, and DPA release delay.     

Peptide nucleic acids: deoxyribonucleic acid mimics with a peptide backbone

This tutorial briefly describes a new class of synthetic biopolymer, which is referred to as peptide nucleic acid (PNA). In PNA, individual nucleobases are linked to an achiral neutral peptide backbone. PNA exhibits the hybridization characteristic (e.g., Watson—Crick duplex formation) of DNA. The achiral peptide backbone provides similar interbase distances as natural DNA, and adequate flexibility to permit base pair interactions with complementary RNA or DNA strands. Several potential applications of PNA oligomers in biotechnology are suggested. These include the use of PNAs as a probe for specific recognition of a DNA or RNA sequence selective, purification of nucleic acids via designed high affinity binding to PNA, screening for DNA mutations, and as possible therapeutic agents.