Induction of mutation in phage T4 by extracellular treatment with methylene blue and visible light

Summary Photodynamic inactivation with methylene blue and light (MB+Li) of maximally sensitized phages T4 and T4rII nearly followed single-hit kinetics. Not so mutation induction, which in one phage (T4rII N₁₇) tested showed multi-hit kinetics. This difference and the fact that T4rII phages with a GC base pair at the site of the rII-mutation showed no enhanced backmutation when compared to rII phages with an AT base pair or to sign mutants suggests that there is no guanine specificity for MB+Li-induced mutation in phage T4.     

N-methyl-N'-nitro-N-nitrosoguanidine and hydroxylamine induced mutants of the rII region of phage T4

rII mutations of bacteriophage T₄ were induced by in vivo treatment with N-methyl-N′-nitro-N-nitrosoguanidine (NG) and in vitro treatment with hydroxylamine (HA). All the NG induced mutations were mappable to small segments of the rIIA cistron and all except one were also highly revertible by 2-aminopurine (AP) treatment. From these observations, it is concluded that treatment of T₄ with NG induces only transitions and contrary to its effects on E. coli, in T₄, NG does not induce any deletions. Spectra of HA and NG induced mutants of the rIIA cistron were compared. Both mutagens seem to be more effective in inducing mutations nearer the two extremities of this cistron and very few in the middle. This asymmetric effect has been seen to be more pronounced in case of NG than in the case of HA.     

Binding of T4 endonuclease V to deoxyribonucleic acid irradiated with ultraviolet light

Endonuclease V of bacteriophage T4 binds to UV-irradiated deoxyribonucleic acid (DNA) but not to unirradiated DNA. We have developed an assay to detect this binding, based on the retention of enzyme--DNA complexes on nitrocellulose filters. The amount of complex retained, ascertained by using radioactive DNA, is a measure of T4 endonuclease V activity. The assay is simple, rapid, and specific, which makes it useful for detecting T4 endonuclease V activity both in crude lysates and in purified preparations. We have used it to monitor enzyme activity during purification and to study binding of the enzyme to DNA under conditions that minimize the ability of the enzyme to nick DNA. From our data we conclude that (1) T4 endonuclease V binds to UV-irradiated DNA but not to DNA that has been previously incised by the endonuclease, (2) equilibrium between the free and complexed form of the enzyme is attained under our reaction conditions, (3) dissociation of enzyme--DNA complexes is retarded by sodium cyanide, and (4) retention of enzyme--DNA complexes on nitrocellulose filters is enhanced by high concentrations of saline--citrate.     

Dictamnine,an alkaloid which crosslinks DNA in the presence of ultraviolet light

In the presence of ultraviolet light, the furoquinoline alkaloid, dictamnine, caused calf thymus DNA to become easily renaturable. The effect was less pronounced than for the furocoumarin, 8-methoxypsoralen. Ease of renaturation is evidence of the formation of interstrand crosslinks in DNA. The mechanism of crosslink formation by this alkaloid may be like that of 8-methoxypsoralen.     

Nature of tryptophan photooxidation products in lysozyme in the presence of methylene blue]

One out of six trytophan residues in two lysozyme modification, obtained under lysozyme photooxidation in the presence of methylene blue, is found to be oxidized to N'-formylkinurenine (in one modification) and to kinurenine (in the other modification). The transition of one modification into another via detaching of N'-formyl group by soft acid hydrolysis has shown that one and the same tryptophan residue is oxidized in both products, Possible mechanism of tryptophan oxidation to the products mentioned is discu-sed on the basis of the hypothesis on signlet mechanism of lysozyme photooxidation in the presence of methylene blue.     

Distribution and characterization of mutations induced by nitrous acid or hydroxylamine in the intron-containing thymidylate synthase gene of bacteriophage T4

The detailed distribution and characterization of 51 hydroxylamine (HA)-induced and 59 nitrous acid (NA)-induced mutations in the intron-containing bacteriophage T4 thymidylate synthase (td) gene is reported here. Mutations were mapped in 10 regions of thetd gene by recombinational marker rescue using plasmid or M13 subclones of thetd gene. Phage crosses using deletion mutants with known breakpoints in the 3′ end of thetd intron subdivided HA and NA mutations which mapped in this region. At least 31 of the mutations map within the 1-kb group I self-splicing intron. Intron mutations mapped only in the 5′ and 3′ ends of the intron sequence, in accordance with the hypothesis that the 5′ and 3′ domains of the T4td intron are essential for correct RNA splicing. RNA sequence analysis of a number of mappedtd mutations has identified two intron nucleotides and one exon nucleotide where both HA- and NA-induced mutations commonly occur. These three loci are characterized by a GC dinucleotide, with the mutations occurring at the cytosine residue. Thus, these data indicate at least three potential sites of both HA- and NA-induced mutagenic hotspot activity within thetd gene.     

Hydrogen peroxide and hydroxyl radical formation by methylene blue in the presence of ascorbic acid

Summary Using ESR we have demonstrated the formation of the ascorbate free radical from sodium ascorbate, methylene blue and light. In oxygen uptake experiments we have observed the production of hydrogen peroxide while spin trapping experiments have revealed the iron catalyzed production of the hydroxyl free radical in this system. The presence of this highly reactive radical suggests that it could be the radical that initiates free radical damage in this photodynamic system.     

Mutation of bacteriophage T4 restoring phage supressor psul+ activity in bacterial strain Escherichia coli BN]

The mutant of bacteriophage T4psu1+XF2 carrying a mutational aleration in the central region of proline-serine tRNA precursor is isolated. The mutational alteration results in the recovery of amber suppressor activity of phage psu1+ serine tRNA in Escherichia coli BN in which the synthesis of this tRNA is normally blocked. Since the amber suppressor activity of mutant serine tRNA becomes sensitive to a restrictive action of strR mutations, its structure seems to be different from that of parental suppressor serine tRNA.     

Escherichia coli Fpg protein and UvrABC endonuclease repair DNA damage induced by methylene blue plus visible light in vivo and in vitro.

pBR322 plasmid DNA was treated with methylene blue plus visible light (MB-light) and tested for transformation efficiency in Escherichia coli mutants defective in either formamidopyrimidine-DNA glycosylase (Fpg protein) and/or UvrABC endonuclease. The survival of pBR322 DNA treated with MB-light was not significantly reduced when transformed into either fpg-1 or uvrA single mutants compared with that in the wild-type strain. In contrast, the survival of MB-light-treated pBR322 DNA was greatly reduced in the fpg-1 uvrA double mutant. The synergistic effect of these two mutations was not observed in transformation experiments using pBR322 DNA treated with methyl methanesulfonate, UV light at 254 nm, or ionizing radiation. In vitro experiments showed that MB-light-treated pBR322 DNA is a substrate for the Fpg protein and UvrABC endonuclease. The number of sites sensitive to cleavage by either Fpg protein or UvrABC endonuclease was 10-fold greater than the number of apurinic-apyrimidinic sites indicated as Nfo protein (endonuclease IR)-sensitive sites. Seven Fpg protein-sensitive sites per PBR322 molecule were required to produce a lethal hit when transformed into the uvrA fpg-1 mutant. These results suggest that MB-light induces DNA base modifications which are lethal and that these modifications are repaired by Fpg protein and UvrABC endonuclease in vivo and in vitro. Therefore, one of the physiological functions of Fpg protein might be to repair DNA base damage induced by photosensitizers and light.     

The induction and repair of DNA damage detected by the DNA precipitation assay in Chinese hamster ovary cells treated with acridine orange + visible light

The photodynamic effect of the dye acridine orange (AO) in combination with visible light (400-700 nm) was studied in Chinese hamster ovary (CHO) cells, the endpoints investigated being induction, as well as repair, of DNA strand breaks. Cells were treated for 20 min with AO (0.1-3.0 micrograms/ml), washed free of excess dye and subsequently exposed to low doses of visible light (2 x 40 W/8 W/m2) for 5-15 min. AO proved to be an efficient sensitizer for light-induced DNA strand breaks, detected with the DNA precipitation assay, and expressed as percentage of DNA precipitated. The induction of breaks was linear up to 0.5 micrograms/ml AO + 10 min of light, which corresponds to 55% precipitated DNA, and was dependent on the concentration of AO as well as on the dose of light delivered. As a comparison, 18 Gy of X-rays was required to yield an equivalent amount of induced DNA strand breaks. The rejoining of the light-induced DNA strand breaks was studied by incubating the AO-sensitized cells for 30-120 min at 37 degrees C directly after light exposure. A fast recover of 67-91% of the damage (compared to initial damage, recovery time = 0, and dependent on the concentration of AO) was observed during the first 30 min of incubation. However, a significant amount of DNA damage remained after 2 h of recovery. These remaining, long-lived lesions might be involved in the photoinduced and acridine-sensitized chromosomal aberrations and sister-chromatid exchanges (SCE). The significance of these observations is discussed in relation to AO-sensitized and photoinduced DNA damage and chromosomal alterations.