Ultraviolet Sensitivity of the Biological Activity of ΦX174 Virus, Single-Stranded DNA, and RF DNA

Action spectra for inactivation of varphiX virus, free varphiX single-stranded DNA, and double-stranded varphiX DNA (RF) have been measured using light of wavelength 225-302 mmu. The sensitivity of RF has been determined using bacterial hosts both capable and incapable of reactivation of UV damage. The inactivation of varphiX virus is due, at all wavelengths, to damage to its DNA; it appears that, below 240 mmu, energy absorbed by viral structural protein may inactivate the viral DNA. The variation of the probability of inactivation by an absorbed quantum (quantum yield) with wavelength, in the case of free-single-stranded varphiX DNA, suggests that energy absorbed by pyrimidine residues is more likely to yield inactivation than absorption by purines. This implies that energy transfer is not so extensive as to make all absorbed energy available to pyrimidines.     

Features of the damage produced by proflavine on transforming deoxyribonucleic acid.

Proflavine formed a complex with transforming deoxyribonucleic acid (DNA) from Haemophilus influenzae, with optimal formation at a ratio of proflavine to DNA of 0.06. The rate of dissociation of the complex by dialysis increased in the order: native, denatured, renatured DNA. The transforming activity of the DNA was reduced by its interaction with proflavine. This inactivation was dependent on the physical state of the DNA, the proflavine concentration, and the temperature. DNA that had been denatured and renatured was most sensitive; native DNA was much less sensitive. The inactivation remained after dialysis and was stable to prolonged storage. It is concluded that the inactivation of transforming DNA by proflavine takes place by a mechanism different from that of DNA-proflavine complex formation.     

Effects of Oxygen Radical Scavengers on the Inactivation of SS ΦX174 DNA by the Semi-Quinone Free Radical of the Antitumor Agent Etoposide

We have studied the effects of oxygen radical scavengers on the inactivation of ss ΦX174 DNA by the semi-quinone free radical of the antitumor agent etoposide (VP 16-213), which was generated from the ortho-quinone of etoposide at pH ≥ 7.4. A semi-quinone free radical of etoposide is thought to play a role in the inactivation of ss ΦDX174 DNA by its precursors 3',4'-ortho-quinone and 3',4'-ortho-dihydroxy-derivative. The possible role of oxygen radicals formed secondary to semi-quinone formation in the inactivation of DNA by the semi-quinone free radical was investigated using the hydroxyl radical scavengers t-butanol and DMSO. the spin trap DMPO, the enzymes catalase and superoxide dismutase, the iron chelator EDTA and potassium superoxide. Hydroxyl radicals seem not important in the process of inactivation of DNA by the semi-quinone free radical, since t-butanol, DMSO, catalase and EDTA had no inhibitory effect on DNA inactivation. The spin trapping agent DMPO strongly inhibited DNA inactivation and semi-quinone formation from the ortho-quinone of etoposide at pH ≥ 7.4 with the concomitant formation of a DMPO-OH adduct. This adduct probably did not arise from OH· trapping but from trapping of O₂^-. DMSO increased both the semi-quinone formation from and the DNA inactivation by the ortho-quinone of etoposide at pH ≥ 7.4. Potassium superoxide also stimulated ΦDX174 DNA inactivation by the ortho-quinone at pH ≤ 7. From the present study, it is also concluded that superoxide anion radicals probably play an important role in the formation of the semi-quinone free radical from the orthoquinone of etoposide, thus indirectly influencing DNA inactivation.     

Early events after infection of Escherichia coli by bacteriophage T5. II. Control of the bacteriophage-induced 5'-nucleotidase activity.

The control of activity of the bacteriophage T5-induced 5'-nucleotidase is dependent upon the amount of T5 parental DNA injected into the cell and expressed. When only the first-step transfer DNA is injected and expressed the amount of 5'-nucleotidase activity observed is two to three times the maximum amount observed after normal T5 infection, and inactivation of the enzyme does not occur. Enzyme inactivation occurs only after the remaining DNA is injected, but only limited expression of this DNA is required. The control of the nucleotidase inactivation process is similar to that for the repair of the nicks in parental DNA, and is probably mediated by a class IIa protein.     

Photodynamic Action on Native and Denatured Transforming Deoxyribonucleic Acid from Haemophilus influenzae

The photodynamic inactivation of native or denatured transforming deoxyribonucleic acid (DNA) from Haemophilus influenzae is described. The inactivation at the same pH was higher for denatured than native DNA. At acidic pH, the inactivation both for native and denatured DNA was faster than at alkaline pH. The guanine content of photoinactivated native DNA at neutral pH was less than untreated DNA. The inactivation of biological activity was more extensive than the alteration of guanine. The absorption spectrum of photoinactivated native or denatured DNA was only slightly different than the control DNA at the different experimental conditions.     

Inactivation of de novo DNA methyltransferase activity by high concentrations of double-stranded DNA

The activity of eukaryotic DNA methyltransferase diminishes with time when the enzyme is incubated with high concentrations (200-300 micrograms/ml) of unmethylated double-stranded Micrococcus luteus DNA. Under similar conditions, single-stranded DNA induces only a limited decrease of enzyme activity. The inactivation process is apparently due to a slowly progressive interaction of the enzyme with double-stranded DNA that is independent of the presence of S-adenosyl-L-methionine. The inhibited enzyme cannot be reactivated either by high salt dissociation of the DNA-enzyme complex or by extensive digestion of the DNA. Among synthetic polydeoxyribonucleotides both poly(dG-dC).poly(dG-dC) and poly(dA-dT).poly(dA-dT), but not poly(dI-dC).poly(dI-dC), cause inactivation of DNA methyltransferase. This inactivation process may be of interest in regulating the 'de novo' activity of the enzyme.     

Mechanism of dehydration inactivation of Lactobacillus plantarum

The mechanism of dehydration inactivation of Lactobacillus plantarum cells after vacuum-drying above saturated salt solutions was studied. The method used is based on the hypothesis that DNase diffuses into cells with damaged cell membranes/walls and hydrolyses the intracellular DNA. Intact, undamaged cells and cells inactivated by either dehydration or heat treatent were incubated in the presence of DNase. The release of DNA hydrolysis products into the incubation medium was measured. It was shown that dehydration inactivation of L. plantarum, but not thermal inactivation, was associated with clear evidence of membrane damage. The residual glucose-fermenting activity of the dehydrated cells related to the release of hydrolysed DNA in the medium, but there was no such relationship with heat-treated cells. Addition of sorbitol to cells before dehydration increased the residual glucose-fermenting activity after drying and this was associated with a reduced rate of DNA hydrolysis. It is concluded that cell wall and/or cell membrane damage is an important mechanism of dehydration inactivation, but that thermal inactivation (up to 60°C) occurs by a different mechanism.Correspondence to: K. van't Riet     

Loss of activity of transforming deoxyribonucleic acid after uptake by Haemophilus influenzae

Voll, Mary Jane (University of Pennsylvania, Philadelphia), and Sol H. Goodgal. Loss of activity of transforming deoxyribonucleic acid after uptake by Haemophilus influenzae. J. Bacteriol. 90:873-883. 1965.-Transforming deoxyribonucleic acid (DNA) which has been irreversibly removed from solution by competent cells undergoes a progressive loss in marker activity when tested by lysis of the cells and exposure to new recipient cells. The loss of activity is limited and marker-specific, with greater inactivation of those markers with lower efficiencies of transformation. Recipient factors or donor factors which have undergone recombination, as measured by the appearance of linked markers, do not undergo inactivation. The efficiency of transformation can be correlated with the sensitivity of a marker to inactivation after DNA uptake. A mutation which affects the efficiency of transformation is found to increase sensitivity to postuptake inactivation. The rate of inactivation is temperature-dependent. At temperatures of 20 and 45 C, marker inactivation can occur without concomitant recombination. During the uptake process, DNA is retained in an acid-insoluble form, indicating that the fate of Haemophilus influenzae DNA differs from the fate of transforming DNA in pneumococcus.     

Affinity modification of DNA polymerase I from Escherichia coli and its Klenow fragment with nucleotide imidazolides

Affinity modification of E. coli DNA polymerase I and its Klenow fragment by imidazolides of dNMP (Im-dNMP) and dNTP was studied. DNA polymerase activity of DNA polymerase I was reduced by both Im-dNMP and Im-dNTP. However Im-dNTP does not inactivate of the Klenow fragment. The level of covalent labelling of both enzymes by radioactive Im-dNTP did not exceed 0.01 mol of reagent per mol of enzyme. But the deep inactivation of DNA polymerase I by Im-dNTP was observed. It is likely that this inactivation is due to the formation of intramolecular ether followed by phosphorylation of the carboxyl group. This assumption is strongly supported by the increase of the isoelectrical point of DNA polymerase I after its incubation with Im-dNTP in conditions of enzyme inactivation. All data permit us to suggest that the affinity modification of both enzymes by Im-dNMP and covalent labeling by Im-dNTP takes place without complementary binding of dNTP moiety with the template. However inactivation of DNA polymerase I by Im-dNTP occurs only if the dNTP-moiety is complementary to the template in the template.primer complex. It was shown that His residue was phosphorylated by Im-dNMP and Tyr or Ser residues between Met-802 and Met-848 were phosphorylated by Im-dNTP. We suppose that there are two states of DNA polymerase active site for the binding of dNTPs. One of them is independent on the template, in the other state the dNTP hydrogen bond with the template is formed.     

Determination of pyrimidine dimers in Escherichia coli and Cryptosporidium parvum during UV light inactivation, photoreactivation, and dark repair

UV inactivation, photoreactivation, and dark repair of Escherichia coli and Cryptosporidium parvum were investigated with the endonuclease sensitive site (ESS) assay, which can determine UV-induced pyrimidine dimers in the genomic DNA of microorganisms. In a 99.9% inactivation of E. coli, high correlation was observed between the dose of UV irradiation and the number of pyrimidine dimers induced in the DNA of E. coli. The colony-forming ability of E. coli also correlated highly with the number of pyrimidine dimers in the DNA, indicating that the ESS assay is comparable to the method conventionally used to measure colony-forming ability. When E. coli were exposed to fluorescent light after a 99.9% inactivation by UV irradiation, UV-induced pyrimidine dimers in the DNA were continuously repaired and the colony-forming ability recovered gradually. When kept in darkness after the UV inactivation, however, E. coli showed neither repair of pyrimidine dimers nor recovery of colony-forming ability. When C. parvum were exposed to fluorescent light after UV inactivation, UV-induced pyrimidine dimers in the DNA were continuously repaired, while no recovery of animal infectivity was observed. When kept in darkness after UV inactivation, C. parvum also showed no recovery of infectivity in spite of the repair of pyrimidine dimers. It was suggested, therefore, that the infectivity of C. parvum would not recover either by photoreactivation or by dark repair even after the repair of pyrimidine dimers in the genomic DNA.     

Targeting of >1.5 Mb of Human DNA into the Mouse X Chromosome Reveals Presence of cis-Acting Regulators of Epigenetic Silencing

Regulatory sequences can influence the expression of flanking genes over long distances, and X chromosome inactivation is a classic example of cis-acting epigenetic gene regulation. Knock-ins directed to the Mus musculus Hprt locus offer a unique opportunity to analyze the spread of silencing into different human DNA sequences in the identical genomic environment. X chromosome inactivation of four knock-in constructs, including bacterial artificial chromosome (BAC) integrations of over 195 kb, was demonstrated by both the lack of expression from the inactive X chromosome in females with nonrandom X chromosome inactivation and promoter DNA methylation of the human transgene in females. We further utilized promoter DNA methylation to assess the inactivation status of 74 human reporter constructs comprising >1.5 Mb of DNA. Of the 47 genes examined, only the PHB gene showed female DNA hypomethylation approaching the level seen in males, and escape from X chromosome inactivation was verified by demonstration of expression from the inactive X chromosome. Integration of PHB resulted in lower DNA methylation of the flanking HPRT promoter in females, suggesting the action of a dominant cis-acting escape element. Female-specific DNA hypermethylation of CpG islands not associated with promoters implies a widespread imposition of DNA methylation during X chromosome inactivation; yet transgenes demonstrated differential capacities to accumulate DNA methylation when integrated into the identical location on the inactive X chromosome, suggesting additional cis-acting sequence effects. As only one of the human transgenes analyzed escaped X chromosome inactivation, we conclude that elements permitting ongoing expression from the inactive X are rare in the human genome.     

Inhibition of short patch and long patch base excision repair by an oxidized abasic site

5'-(2-Phosphoryl-1,4-dioxobutane) (DOB) is an oxidized abasic lesion that is produced by a variety of DNA damaging agents, including several antitumor antibiotics. DOB efficiently and irreversibly inhibits DNA polymerase β, an essential base excision repair enzyme in mammalian cells. The generality of this mode of inhibition by DOB is supported by the inactivation of DNA polymerase λ, which may serve as a possible backup for DNA polymerase β during abasic site repair. Protein digests suggest that Lys72 and Lys84, which are present in the lyase active site of DNA polymerase β, are modified by DOB. Monoaldehyde analogues of DOB substantiate the importance of the 1,4-dicarbonyl component of DOB for efficient inactivation of Pol β and the contribution of a freely diffusible electrophile liberated from the inhibitor by the enzyme. Inhibition of DNA polymerase β's lyase function is accompanied by inactivation of its DNA polymerase activity as well, which prevents long patch base excision repair of DOB. Overall, DOB is highly refractory to short patch and long patch base excision repair. Its recalcitrance to succumb to repair suggests that DOB is a significant source of the cytotoxicity of DNA damaging agents that produce it.     

Fate of transforming bacteriophage HP1 deoxyribonucleic acid in Haemophilus influenzae lysogens.

The biological fate of temperate phage HP1 deoxyribonucleic acid (DNA) was followed after uptake by defectively lysogenic competent Haemophilus influenzae cultures. The similar inactivation kinetics of three single phage genetic markers and of their triple combination indicated a complete rather than partial destruction of about half of the adsorbed DNA molecules. Intracellular DNA breakdown products were tentatively identified by hydroxyapatite column chromatography as short single strands and extensively damaged short double strands. Integrated donor DNA (after single-strand insertion?) was still highly efficient for triple-marker co-transformation. This suggests that whole or nearly whole donor DNA molecules were integrated. Some donor DNA was never integrated but remained largely unaltered. This DNA fraction did not contain significant amounts of recipient prophage marker activity. It is concluded that it had not participated in some kind of reciprocal recombination event involving the recipient chromosome. Since very similar phage DNA marker inactivation rates were observed after adsorption by competent nonlysogenic recipients (transfection), the relationship between biological inactivation of adsorbed donor phage DNA and its integration in lysogenic recipients is not clear.     

Critical DNA damage and mammalian cell reproduction

Synchronous Chinese hamster cells accumulated ¹²⁵I-induced DNA damage in the G₂ + M2 period at 4 °C. The position of the ¹²⁵I within the nuclear DNA was varied by incorporating [¹²⁵I]iododeoxyuridine at various times during the previous DNA replication period. When the initial cell inactivation efficiency was compared for damage accumulated in various regions of nuclear DNA, it was found that the efficiency of inactivation was least in early replicating DNA, and that it gradually increased, reaching a maximum during the fourth and fifth hours of the six-hour DNA replication period. Because the DNA that replicated during this maximum corresponds to that DNA which later forms centromeric and near-centromeric regions of the chromosomes, damage in centromeric region DNA may be critical in causing mammalian cell inactivation.     

Mechanism of Inactivation of Bacteriophage J1 by Glutathione

Mechanism of inactivation of a double-stranded DNA phage, phage Jl of Lactobacillus casei, by reduced form of glutathione (GSH) was studied.

Air (oxygen) bubbling, oxidizing agents and transition metal ions enhanced the rate of inactivation of the phage by GSH. Partial oxidation of GSH resulted in a more rapid rate of inactivation. In contrast, nitrogen bubbling, reducing agents, chelating agents and radical scavengers prevented the inactivation. Fully oxidized GSH had no phagocidal effect. These results indicate that the inactivating effect of GSH requires the presence of molecular oxygen and is caused by free radical involved in the mechanism of GSH oxidation.

The target of GSH in the phage particle was not the tail protein but DNA. GSH reacted with phage DNA and caused single-strand scissions in the DNA, as exhibited by alkaline sucrose gradient centrifugation; thus inactivating phage.     

DNA core ionization and cell inactivation

Whether inner-shell ionizations of DNA atoms, called core ionizations, are critical events for cell inactivation by ionizing radiations such as 100 keV electrons and gamma rays has been investigated. The number of core ionizations in DNA atoms per gray of the two types of radiations is calculated from various Monte Carlo track simulations. The probability that a core ionization leads to cell inactivation is deduced from experimental values of the RBEs of ultrasoft X rays. The contribution to V79 cell inactivation solely due to the core ionizations in DNA is found to be 75 +/- 27% for energetic electrons and gamma rays. This surprisingly large contribution strongly suggests the presence of new mechanisms associated with critical lesions for cell inactivation.     

Constitutively active DNA damage checkpoint pathways as the driving force for the high frequency of p53 mutations in human cancer

If the major function of the p53 protein is to function as a DNA damage checkpoint protein, then it is reasonable to hypothesize that its inactivation in human cancer must be related to its DNA damage checkpoint function. This hypothesis further implies that in tumor cells one or more of the DNA damage checkpoint pathways has been activated. Otherwise, p53 would not be active and there would be no selective pressure for TP53 mutations. I make the argument that tumorigenic transformation is intrinsically associated with formation of DNA DSBs in every cell cycle leading to activation of DNA damage checkpoint pathways. In turn, activation of the DNA DSB checkpoint provides the selective pressure for the high frequency of p53 inactivation in human cancer.     

Ultraviolet Inactivation Dichroic Ratio of Oriented fd Bacteriophage

Polarized UV light irradiation of flow-oriented fd bacteriophage indicates that the degree of damage (inactivation) depends on the relative orientation of the light polarization vector and the plane of the DNA bases. The technique of anisotropic UV inactivation was evaluated, and further information on the orientation in this virus was gained. The fd bacteriophage were aligned and irradiated with plane-polarized monochromatic UV light either parallel or perpendicular to the virus axis. Variation of the inactivation dichroic ratio with wavelength implicated virus inactivation by light absorbed in both the DNA and protein. Analysis of the wavelength variation of inactivation dichroic ratios gave molecular dichroic ratios of 0.76 and 1.48 for the DNA and protein components, respectively. On the basis of these anisotropic inactivation studies, the average angle of DNA base tilt in fd was calculated to be 29-32°, a value in agreement with the absorption dichroism studies of Bendet and Mayfield.