Joh. Blok

Contribution of various types of damage to inactivation of a biologically-active double-stranded circular DNA by gamma-radiation.

SummaryThe double-stranded circular DNA of the bacteriophage PM2 has been irradiated in oxygenated solution by 60Co gamma rays. The following quantities have been determined as a function of dose: The average number of single-strand breaks and that of double-strand breaks per molecule, the biological activity of the irradiated sample as such, the biological activity after mild denaturation (to denature molecules containing a break) and the biological activity after denaturation and membrane filtration (i.e. the biological activity of DNA without single- and double-strand breaks.)From the collected data it can be deduced that 4·5 ± 0·5 per cent of the inactivation is a consequence of double-strand breaks, 8·5 ± 4·2 per cent of single-strand breaks and 87·0 ± 4·2 per cent of nucleotide damage. Only about 2 per cent of the single-strand breaks is lethal, whereas the efficiency of inactivation of nucleotide damage is about 30 per cent of the nucleotide damage which is lethal if present in single-stranded DNA....

Reaction rate of OH radicals with phi X174 DNA: influence of salt and scavenger.

A derivation is given for the dependence of the rate constant of the reaction of OH radicals with a spherical macromolecule on the rate by which such radicals are scavenged by the medium. Experiments were carried out with oxygenated solutions of dilute single-stranded phi X174 DNA at 10(-4)M NaCl (large reaction radius of DNA) or at 10(-4)M NaCl + MgCl2 (small reaction radius) with t-butanol as a scavenger. The results of these experiments cannot be described by simple second-order competition, but can be explained by the predicted dependence of the rate constant of the reaction OH + DNA on the concentration of t-butanol. Furthermore, the results show that only part of the reactions of OH radicals with phi X174 DNA leads to DNA inactivation, and that even at zero scavenger concentration OH radicals are scavenged by other molecules than DNA, presumably impurities remaining even after careful purification of the DNA.

Binding Stoichiometry of the Gene 32 Protein of Phage T4 in the Complex with Single Stranded DNA Deduced from Boundary Sedimentation

Short 145 base DNA fragments in complex with the helix destabilizing protein of bacteriophage T4, GP32, have been studied with boundary sedimentation. The sedimentation coefficient was determined as a function of concentration, protein-nucleic acid ratio, temperature and salt concentration. It can be concluded that the measured values reflect the properties of the saturated DNA-GP32 complex. A combination of the earlier obtained translational diffusion coefficient of the complex with the sedimentation coefficient yields its anhydrous molecular weight (Mw = 5.4.10(5) D), which corresponds to a size of the binding site of 10 nucleotides per protein. This procedure is not sensitive to the presence of non-binding protein molecules and to the assumed protein concentration, and therefore, it seems more reliable than a determination from titration experiments. Similar sedimentation measurements were performed with tRNA-complexes containing 76 nucleotides. The translational diffusion coefficient can be calculated from the measured rotational diffusion coefficient and assuming the same hydrodynamic diameter for this complex as obtained for the 145 b DNA complex. The molecular weight derived from the data then also leads to a binding site size of about 10 nucleotides. This suggests that also the short tRNA-complex forms an open, strongly solvated structure, as was proposed for the 145 b DNA-GP32 complex.

THE INDUCTION OF MUTATIONS BY IONIZING RADIATION IN BACTERIOPHAGE /phi/X174 AND IN ITS PURIFIED DNA

Abstract By X-irradiation of a mutant of the Escherichia coli bacteriophage φX 174 , with a more extended host-range than the wild type, back mutations could be induced. During irradiation the bacteriophage protein was protected against indirect effects by means of thiourea. The mutagenic efficiency was about 2 · 10 −4 per lethal hit. The same type of mutation was found in the progeny produced by irradiated DNA in bacterial spheroplasts. In these experiments the DNA was separated from the phage protein by extraction with phenol, and irradiated in aqueous solution at a concentration of 5 μg/ml. Under these conditions the effect appeared to be due to radicals produced in the water since the inactivation dose was very small, namely only about 40 rad in very pure preparations. The number of mutants produced per lethal hit was insignificant in the presence of oxygen, but rose to 2 · 10 −4 when oxygen was replaced by hydrogen.

The indirect action of gamma-rays on the coat protein of bacteriophage particles.

SummarySuspensions of bacteriophage T4 in 25 mM phosphate buffer, pH 7·1, were irradiated with 60Co γ-rays. Measurements of the viscosity of irradiated samples before and after treatment with DNA-ase and trypsin showed that DNA is released from damaged phage particles. The phage debris acts as an effective radical scavenger and at higher doses protects the residual phage, thereby decreasing the inactivation rate. In irradiated suspensions of T4 and ΦX174 phage particles biologically-active subviral particles able to infect bacterial spheroplasts were found. Sedimentation in a sucrose gradient showed that the subviral particles of T4 have nearly the same sedimentation coefficient as unirradiated particles. Examination of irradiated samples with the electron microscope revealed that they contained many particles with the sheath contracted. The subviral particles produced by irradiation resemble those obtained with urea but do not show the tail clusters found in samples treated with urea.

The influence of some sulphydryl compounds on γ-ray-induced inactivation and reversion to prototrophy in E. coli B fil- citrul-

Abstract A study was made of the influence of cysteamine (β-mercaptoethylamine), S ,2-aminoethylisothiourea, monothioglycol (β-mercaptoethanol) and cysteine on inactivation and mutation induction in Escherichia coli B fil- citrul- irradiated in nitrogen with 60 Co γ-rays. All the compounds afforded protection against inactivation, but when they were added immediately before irradiation no protection was observed below about 10 krad. After 20 min pre-irradiation incubation with the compounds only cysteamine showed this effect. All the compounds appeared to protect against inactivation and against mutation induction to the same extent.

γ-Rays Inactivate ϕX174 DNA in Frozen Anoxic Solutions at −20°C Mainly by Reactions of Dry Electrons

SummaryBacteriophage ϕX174 DNA irradiated at −20°C in rapidly frozen anoxic solution is not inactivated by the well known water radicals but most probably chiefly by reactions of dry electrons.