Sequence analysis of ultraviolet-induced mutations in M13lacZ hybrid phage DNA

We have studied the specificity of ultraviolet (u.v.) mutagenesis in single-stranded DNA phage by analyzing u.v.-induced forward mutations in the lac insert of M13mp2 hybrid phage. Sequence analysis of 114 lac mutants derived from u.v.-irradiated phage grown in u.v.-irradiated cells showed that ultraviolet induces mainly single-nucleotide substitutions and deletions in progeny phage DNA. A total of 74% of the single-base substitution mutations occurred at sites of adjacent pyrimidines in the single-stranded DNA, with both T----C and C----T transitions predominating in the u.v. spectrum. Single-nucleotide deletion mutations occurred preferentially in tracts of repeated pyrimidine nucleotides. Tandem, double-base substitutions did not represent a major class of u.v.-induced mutations, but nearly 10% of mutant clones contained multiple, non-tandem nucleotide changes.     

Bromouracil mutagenesis in Escherichia coli evidence for involvement of mismatch repair.

Summary Bromouracil mutagenesis was studied in several strains of E. coli in combination with measurement of incorporation of bromouracil in DNA. For levels below 10% total replacement of bromouracil for thymine, mutagenesis was negligible compared with higher levels of incorporation. Such a nonlinear response occurred both when the bromouracil was evenly distributed over the genome and when a small proportion of the genome was highly substituted. Also, the mutation frequency could be drastically lowered by amino acid starvation following bromouracil incorporation. These observations suggest the involvement of repair phenomena. Studies of mutagenesis in recA ^– and uvrA ^– mutants, as well as studies of prophage induction, did not support an error prone repair pathway of mutagenesis. On the other hand, uvrD ^– and uvrE ^– mutants, which are deficient in DNA mismatch repair, had much increased mutation frequencies compared with wild type cells. The mutagenic action of bromouracil showed specificity under the conditions used, as demonstrated by the inability of bromouracil to revert an ochre codon that was easily revertable by ultraviolet light irradiation. The results are consistent with a mechanism of bromouracil mutagenesis involving mispairing, but suggest that the final mutation frequencies depend on repair that removes mismatched bases.     

Differential effects of procaine and phenethyl alcohol on excision repair of DNA in u.v.-irradiated Escherichia coli

Experiments were performed to investigate the involvement of the cell membrane in the excision DNA repair process in Escherichia coli. Two membrane-binding drugs, procaine and phenethyl alcohol (PEA), inhibited liquid-holding recovery (LHR) in u.v.-irradiated E. coli wild-type and recA strains. In uvrB and polA strains where, after u.v.-irradiation, LHR was absent the two drugs had no effect. Both drugs markedly reduced the removal of u.v.-induced thymine dimers in the DNA of wild-type cells (H/r30). Analysis by alkaline sucrose gradients revealed that PEA inhibited the incision step in excision repair. In contrast, procaine had no effect on incision but apparently inhibited the late steps in excision repair. PEA dissociated DNA from the cell membrane, whereas procaine did not. The results suggest that the two drugs PEA and procaine inhibit LHR and the excision repair process operating on u.v.-induced damage in E. coli by at least two different mechanisms each of which may involve the cell membrane.     

Near ultraviolet DNA damage induces the SOS responses in Escherichia coli.

The influence of the growth delay induced by near u.v. radiation on the SOS response was monitored by comparing the level of sfiA expression by means of a sfiA::lacZ fusion in both a nuvA+ cell and an isogenic nuvA mutant. The mutant lacks 4-thiouridine in its tRNA and does not exhibit the near u.v.-induced growth delay. Although the two strains exhibit similar sfiA induction levels after 254 nm irradiation, their behaviour is different after illumination with near u.v. light, including solar u.v. Inducibility is 10-20 times higher in the nuvA mutant than in the parent strain. Furthermore, pre-illumination with broad band near u.v. light does not affect the 254 nm-induced sfiA response in the mutant but reduces it by a factor of 3-4 in the parent strain. The kinetics of sfiA induction in near u.v.-illuminated nuvA+ cells, whether treated with 254 nm light or not, is unusual and follows the growth curve: only after 50 min is sfiA derepression observed. It can be concluded that (i) near u.v.-induced DNA lesions are able to trigger the SOS response and (ii) the growth delay effect reduces this response, whether triggered by u.v. or near u.v. light. Hence 4-thiouridine in tRNA acts as a built-in antiphotomutagenic 'device' protecting Escherichia coli cells against mutagenesis and the induction of the SOS response by near u.v. light and sunlight.     

Pyrimidine dimers are not the principal pre-mutagenic lesions induced in lambda phage DNA by ultraviolet light

Experiments were performed to examine the role of cyclobutyl pyrimidine dimers in the process of mutagenesis by ultraviolet (u.v.) light. Lambda phage DNA was irradiated with u.v. and then incubated with an Escherichia coli photoreactivating enzyme, which monomerizes cyclobutyl pyrimidine dimers upon exposure to visible light. The photoreactivated DNA was packaged into lambda phage particles, which were used to infect E. coli uvr- host cells that had been induced for SOS functions by ultraviolet irradiation. Photoreactivation removed most toxic lesions from irradiated phage, but did not change the frequency of induction of mutations to the clear-plaque phenotype. This implies that cyclobutyl pyrimidine dimers can be lethal, but usually do not serve as sites of mutations in the phage. The DNA sequences of mutants derived from photoreactivated DNA showed that almost two-thirds (16/28) were transitions, the same fraction found for u.v. mutagenesis without photoreactivation. These results show that in this system, the lesion inducing transitions (the major type of u.v.-induced mutation) is not the cyclobutyl pyrimidine dimer; a strong candidate for a mutagenic lesion is the Pyr(6-4)Pyo photoproduct. On the other hand, photoreactivation of SOS-induced host cells before infection with u.v.-irradiated phage reduced mutagenesis substantially. In this case, photoreversal of cyclobutyl dimers serves to reduce expression of the SOS functions that are required in the process of targeted u.v. mutagenesis.     

Effect of Bromouracil-containing Deoxyribonucleic Acid on Bacillus subtilis

Gimlin, Dixie M. (Oklahoma State University, Stillwater), Sue D. Hardman, Betty N. Kelley, Grace C. Butler, and Franklin R. Leach. Effect of bromouracil-containing deoxyribonucleic acid on Bacillus subtilis. J. Bacteriol. 92:366-374. 1966.-Replacement of one-half of the thymine with bromouracil in Bacillus subtilis transforming deoxyribonucleic acid (DNA) resulted in a slight decrease in transforming activity, but, when used at high concentrations, this DNA preparation inhibited cell growth. Acid-hydrolyzed DNA, or addition of equivalent concentrations of the free base bromouracil in a transforming mixture, was without effect on cell growth. Treatment of the DNA preparation with deoxyribonuclease completely destroyed transforming activity and killing effect, whereas treatments with ribonuclease and trypsin were without effect on either transformation or killing activity. Growth of competent B. subtilis cells in test tubes was inhibited by high concentrations of both normal and bromouracil-containing DNA, with the bromouracil-containing DNA being significantly more inhibitory. This type of inhibition was also reflected in the time of division of the cells. The inhibitory effect was not due to viscosity, or to mutagenicity. The time course of killing paralleled transformation, and competency was required. These results can be interpreted as being due to uptake of homologous but imperfect DNA (containing bromouracil instead of thymine) by means of the systems involved in transformation, followed by either integration (resulting in lethal transformation, activation of a defective, nonlytic but lethal prophage) or interference with the recombination mechanism.     

Ultraviolet-induced dimerization of non-adjacent pyrimidines. A potential mechanism for the targeted -1 frameshift mutation

The DNA photoproduct responsible for the ultraviolet (u.v.)-induced targeted -1 frameshift mutation is unknown. Based on mutagenesis studies by others, we surmised that this lesion might be found in high abundance in single-stranded DNA. u.v. irradiation of the single-stranded alternating copolymer poly[d(G-T)] yielded a photoproduct that was characterized in detail. It consists of a thymine-thymine cyclobutane dimer of predominantly cis-syn configuration occurring between non-adjacent thymidyl residues on the same strand. Its formation is strongly inhibited in double-stranded DNA. A similar u.v. photoproduct was obtained in higher yield from the polypyrimidine alternating copolymer poly[d(C-T)] under conditions in which it is single-stranded. It is proposed that replication across the unrepaired photoproduct: (formula; see text) is the cause of the targeted u.v.-induced -1 frameshift mutation.     

Effects of extremely low frequency (ELF) electric fields on cell growth and DNA repair in human skin fibroblasts

A number of physical and chemical agents in the environment have been studied for their ability to induce or alter DNA repair mechanisms in human cells. We have investigated the effects of 60 Hz, 1000 V/cm electric fields on DNA repair in normal human fibroblasts in vitro. An examination was done on the ability of electric fields suspected to cause damage which could be repaired by thymine dimer excision and measurable by the bromodeoxyuridine photolysis assay. The thymine dimer assay with enzyme-sensitive site analysis was used to measure the cells' capacity for removing ultraviolet light (u.v.)-induced pyrimidine dimers; during exposure to electric field 24 hr before u.v. irradiation; 24 hr after u.v. irradiation; and up to 48 hr continuously after u.v. irradiation. Cell growth and cell survival following electric field exposure were also studied. Within the limits of these experiments, it was found that exposure to such electric fields did not alter cell growth or survival, and no DNA repair or alteration in DNA excision repair capacity was observed as compared with unexposed control cultures.     

Microinjection of human cell extracts corrects xeroderma pigmentosum defect

Cultured fibroblasts of patients with the DNA repair syndrome xeroderma pigmentosum (XP) were injected with crude cell extracts from various human cells. Injected fibroblasts were then assayed for unscheduled DNA synthesis (UDS) to see whether the injected extract could complement their deficiency in the removal of u.v.-induced thymidine dimers from their DNA. Microinjection of extracts from repair-proficient cells (such as HeLa, placenta) and from cells belonging to XP complementation group C resulted in a temporary correction of the DNA repair defect in XP-A cells but not in cells from complementation groups C, D or F. Extracts prepared from XP-A cells were unable to correct the XP-A repair defect. The UDS of phenotypically corrected XP-A cells is u.v.-specific and can reach the level of normal cells. The XP-A correcting factor was found to be sensitive to the action of proteinase K, suggesting that it is a protein. It is present in normal cells in high amounts, it is stable on storage and can still be detected in the injected cells 8 h after injection. The microinjection assay described in this paper provides a useful tool for the purification of the XP-A (and possibly other) factor(s) involved in DNA repair.     

Changes in the organization of chromosomes during the cell cycle: response to ultraviolet light.

Fusion between mitotic and interphase cells results in the premature condensation of the interphase chromosomes into a morphology related to the position in the cell cycle at the time of fusion. These prematurely condensed chromosomes (PCC) have been used in conjunction with u.v. irradiation to examine the interphase chromosome condensation cycle of HeLa cells. The following observations have been made: (I) There is a progressive decondensation of the chromosomes during G1 which is accentuated by u.v. irradiation: (2) The chromosomes become more resistant to u.v.-induced decondensation during G2 and mitosis. (3) There is a close correlation between the degree of chromosome decondensation and the amount of unscheduled DNA synthesis induced by u.v. irradiation during G1 and mitosis: (4) Hydroxyurea enhances the ability of u.v. irradiation to promote the decondensation of chromosomes during G1, G2 and mitosis. Hydroxyurea also potentiates the lethal action of u.v. irradiation during mitosis and G1. These data are discussed in relation to the suggestion that chromosomes undergo a progressive decondensation during G1 and condensation during G2.     

Excision repair in u.v. (254 nm) damaged non-dividing human skin fibroblasts: a major biological role for DNA polymerase alpha

We have used the eukaryotic DNA polymerase alpha inhibitor, aphidicolin, and the polymerase beta inhibitor, dideoxythymidine, to examine the role of these enzymes in excision repair of ultraviolet (u.v., 254 nm) damage induced in non-dividing (arrested) human skin fibroblasts. The effects of these drugs on u.v.-treated cells have been monitored using a simple and reproducible repair synthesis assay in parallel with viability measurements to determine the degree of inhibition of repair of potentially lethal damage. In agreement with previous studies using density gradients, repair synthesis induced by low fluences of u.v. (less than 3 J m-2) is relatively insensitive to inhibition by aphidicolin compared to high fluences where approximately 85 per cent inhibition is observed at the highest (20 micrograms/ml) aphidicolin concentration employed. However, repair of potentially lethal damage is inhibited by at least 90 per cent over the entire fluence range. Although dideoxythymidine led to considerable inhibition of repair synthesis, the result is probably an artifact under these in vivo conditions. The polymerase beta inhibitor was not toxic to u.v.-treated cells nor did it add to the toxicity of aphidicolin when the drugs were used in combination. We conclude that if the beta polymerase is involved in excision repair then its temporary (4 h) inhibition by dideoxythymidine is entirely reversible. In contrast, polymerase alpha appears to be an enzyme essential to the majority of biologically effective excision repair over the entire u.v. fluence range tested.     

Discrimination between bromouracil and thymine for uptake into DNA in drm- and dra- mutants of Escherichia coli K12.

The relative efficiency of bromouracil and thymine for uptake into DNA was measured in various thymine-requiring strains of Escherichia coli K12. It was found that: 1. Mutants with genotype thyA- dra- discriminate against bromouracil to much greater extent than do mutants with genotype thyA- drm-. 2. The discrimination in dra-mutants is dependent on thymine concentration, whereas discrimination in drm- mutants is almost independent of thymine concentration. It is suggested that the intracellular level of deoxyribose 5-phosphate affects the efficiency of uptake into DNA of bromouracil relative to thymine.     

Plasmid modification of radiation and chemical-mutagen sensitivity in Pseudomonas aeruginosa.

The R factor pMG2 protects Pseudomonas aeruginosa against the lethal effects of ultraviolet (u.v.) and gamma irradiation, and methyl methanesulphonate and N-methyl-N'-nitro-N-nitrosoguanidine treatment. Enhanced survival occurs in strains of uvr+ rec+ (wild-type) genotype and a variety of uvr rec+ type mutants. No protection occurs in a rec A-type mutant. The plasmid also enhances u.v.-induced mutagenesis. These effects appear to be due to host-cell controlled plasmid-determined DNA repair function(s). Studies on P. aeruginosa strains deficient in DNA polymerase I (polyA) suggest that a plasmid-determined repair resynthesis function may be responsible for increased u.v.-survival and enhanced u.v.-mutability in pMG2-containing bacteria.     

Strand breaks and alkali-labile bonds induced by ultraviolet light in DNA with 5-bromouracil in vivo.

Supercircular gamma phage DNA with 10 bromouracils/100 thymine bases, irradiated with 313 nm light in Tris buffer and sedimented on alkaline and neutral gradients, showed 4.6 alkali-labile bonds per true single-strand break, in agreement with Hewitt and Marburger (1975 Photochem. Photobiol. 21:413). The same DNA irradiated in Escherichia coli host cells showed about the same number of breaks in alkaline gradients for equal fluence, but only 0.5 alkali-labile bond per true break. Similarly, E. coli DNA with bromouracil irradiated in the cells showed only 10--20% more breaks when denatured with 0.1 M NaOH than under neutral conditions with 9 M sodium perchlorate at 50 degrees C. These results show that true single-strand breaks occur more frequently than alkali-labile bonds after ultraviolet irradiation of DNA containing bromouracil in cells.     

Direct and indirect effects of ultraviolet light on the mutagenesis of parvovirus H-1 in human cells.

U.v. radiation is directly mutagenic for the single-stranded DNA parvovirus H-1 propagated in human cells. Mutation induction in the progeny of u.v.-irradiated virus increased linearly with the dose and could be ascribed neither to an increased number of rounds of viral replication nor to the indirect activation of an inducible cellular mutator activity by the u.v.-damaged virus. The level of mutagenesis among the descendants of both unirradiated and u.v.-damaged H-1 was enhanced if the host cells had been exposed to sublethal doses of u.v. light before infection. This indirect enhancement of viral mutagenesis in pre-irradiated cells was maximal at multiplicities lower than 0.2 infectious particles/cell. The frequency of mutations resulting from cell pre-irradiation was only slightly higher for u.v.-irradiated than for intact virus. Thus, the induced cellular mutator appeared to be mostly untargeted in the dose range given to the virus. U.v.-irradiation of the cells also enhanced the mutagenesis of u.v.-irradiated herpes simplex virus, a double-stranded DNA virus ( Lytle and Knott , 1982).     

DNA base sequence changes induced by bromouracil mutagenesis of lambda phage

The base sequence changes induced by bromouracil mutagenesis in the cI gene of phage lambda have been determined by direct sequence analysis. Phage DNA mutagenized during prophage replication or during phage lytic growth showed predominantly A · T → G · C transitions. The frequency of this mutation was strongly sequence-dependent: 5′ A-C-G-C 3′ > A-C(A.C or T) > A(A.G or T). The difference in mutability of bases in the gene is not the result of specificity in mutL-dependent mismatch repair, since phage grown in mutL host cells showed the same distribution of bromouracil mutations. The observations made in phage mutagenized with bromouracil in the prophage state should be representative of bromouracil mutagenesis in the Escherichia coli chromosome.     

R plasmids which alter ultraviolet light-sensitivity and enhance ultraviolet light-induced mutability in Pseudomonas aeruginosa.

R plasmids pMG1, R2, R931 and pMG15 increased the survival of Pseudomonas aeruginosa exposed to ultraviolet radiation (u.v.) in the wild type, and uvr and polA mutants but did not alter the u.v.-response of a recA mutant. The R plasmid RPL11 reduced u.v.-survival in the wild type, and uvr and polA mutants but did not alter the u.v.-response of a recA host. All the plasmids enhanced the level of spontaneous and u.v.-induced back mutation (Trp+) in a trpB1 strain. The effect of sublethal concentration of sodium arsenite following u.v.-irradiation was examined. It was concluded that in strains trpB1(pMG1) and trpB1(R931), u.v.-protection is determined by a recA+-dependent, arsenite-sensitive repair pathway, whereas in strains trpB1(R2) and trpB1(pMG15), u.v.-protection is determined by a recA+-dependent, arsenite-insensitive step in DNA repair.     

Relationship between enhanced reactivation and mutagenesis of u.v.-irradiated human cytomegalovirus in normal human cells.

The survival of u.v.-irradiated human cytomegalovirus (HCMV) on u.v.-irradiated human IAFP-1 cells was increased over that on unirradiated cells. Irradiated virus had a higher forward mutation frequency towards temperature sensitivity in irradiated than in unirradiated cells. Enhanced reactivation of u.v.-irradiated HCMV is thus mutagenic in normal human cells. This observation supports the possible induction of an error-prone mode of DNA repair in u.v.-irradiated mammalian cells.     

Inhibition and recovery of DNA synthesis in PHA-stimulated u.v.-irradiated lymphocytes from patients with actinic keratosis

The recovery, after inhibition by u.v.-irradiation, of phytohaemagglutinin-stimulated DNA synthesis is impaired in lymphocytes from patients with multiple actinic keratoses (AK) compared to the recovery in lymphocytes from age-matched, control individuals. This shows that the reduced level (50%) of DNA repair activity in AK ceils is sufficiently different from that in normal cells to significantly affect cellular activity.