Apurinic sites cause mutations in simian virus 40

SV40 has been used as a molecular probe to study the mutagenicity of apurinic sites (Ap) in mammalian cells. Untreated or UV-irradiated monkey kidney cells were transfected with depurinated DNA from the temperature-sensitive tsB201 SV40 late mutant which grows normally at the permissive temperature of 33 degrees C but which is unable to grow at 41 degrees C. Phenotypic revertants were screened at 41 degrees C for their ability to grow at the restrictive temperature and the mutation frequency was calculated in the viral progeny. Ap sites were introduced into DNA by heating at 70 degrees C under acid conditions (pH 4.8). This treatment induces one Ap site per SV40 genome per 15 min of heating as measured by alkaline denaturation or by treatment with the T4-encoded UV-specific endonuclease which possesses Ap-endonuclease activity. The experiments reported here show that Ap sites strongly decrease virus survival with a lethal hit corresponding roughly to 3 Ap lesions per SV40 genome, and indicate for the first time that apurinic sites produced by heating are highly mutagenic in animal cells. UV irradiation of the host cells 24 h prior to transfection with depurinated DNA did not modify the mutation frequency in the virus progeny.     

Relationship Between Prophage Induction and Transformation in Haemophilus influenzae

The interaction between transformation and prophages of HP1c1, S2, and a defective phage of Haemophilus influenzae has been investigated by measurement of (i) the effect of prophage on transformation frequency and (ii) the effect of transformation on phage induction. The presence of any of the prophages does not appreciably alter transformation frequencies in various Rec(+) and Rec(-) strains. However, exposure of competent lysogens to transforming deoxyribonucleic acid (DNA) may induce phage but only in Rec(+) strains, which are able to integrate transforming DNA into their genome. Transformation of Rec(+) lysogens with DNA irradiated with ultraviolet (UV) light causes the production of even more phage than results from unirradiated DNA, but this indirect UV induction is not as effective as direct induction by UV irradiation of lysogens. Both types of UV induction are influenced by the repair capacity of the host. Wild-type cells contain a prophage and can be induced by transformation to produce a defective phage, which kills a small fraction of the cells. Defective phage in wild-type cells are also induced by H. parainfluenzae DNA, and a much larger fraction of the cells is killed. Strain BC200, which is highly transformable but is not inducible for defective phage, is not killed by H. parainfluenzae DNA, suggesting that wild-type cells are killed by killed by this DNA because of phage induction. A minicell-producing mutant, LB11, has been isolated. Some phage induction occurs in this strain when the cells are made competent, unlike the wild type. A large majority of LB11 cells surviving the competence regime are killed by exposure to transforming DNA.     

Unusual properties of a new division mutant of Escherichia coli

The properties of a division mutant of Escherichia coli were investigated. At 42 degrees C, this mutant forms nonseptate, multinucleate, filamentous cells typical of division mutants, and at the permissive temperature, is sensitive to ultraviolet (UV) irradiation. Temperature and UV sensitivities are probably due to a single mutation. The mutant phenotype is dominant to wild type. The mutant cells make DNA nearly as effectively as control cells at 42 degrees C or following UV irradiation. They exhibit normal host-cell reactivation capacities and can express all manifestations of the SOS response with the exception of Weigle reactivation. The genetic lesion which mediates this pleiotropic effect is located very close to the leu locus of the linkage map.     

UV light-induced cyclobutane pyrimidine dimers are mutagenic in mammalian cells.

We used a simian virus 40-based shuttle vector plasmid, pZ189, to determine the role of pyrimidine cyclobutane dimers in UV light-induced mutagenesis in monkey cells. The vector DNA was UV irradiated and then introduced into monkey cells by transfection. After replication, vector DNA was recovered from the cells and tested for mutations in its supF suppressor tRNA marker gene by transformation of Escherichia coli carrying a nonsense mutation in the beta-galactosidase gene. When the irradiated vector was treated with E. coli photolyase prior to transfection, pyrimidine cyclobutane dimers were removed selectively. Removal of approximately 90% of the pyrimidine cyclobutane dimers increased the biological activity of the vector by 75% and reduced its mutation frequency by 80%. Sequence analysis of 72 mutants recovered indicated that there were significantly fewer tandem double-base changes and G X C----A X T transitions (particularly at CC sites) after photoreactivation of the DNA. UV-induced photoproducts remained (although at greatly reduced levels) at all pyr-pyr sites after photoreactivation, but there was a relative increase in photoproducts at CC and TC sites and a relative decrease at TT and CT sites, presumably due to a persistence of (6-4) photoproducts at some CC and TC sites. These observations are consistent with the fact that mutations were found after photoreactivation at many sites at which only cyclobutane dimers would be expected to occur. From these results we conclude that UV-induced pyrimidine cyclobutane dimers are mutagenic in DNA replicated in monkey cells.     

Transformation and mutagenic potential of porphyrin photodynamic therapy in mammalian cells

The transformation and mutagenic potential of porphyrin photodynamic therapy has been examined in mammalian cells. The mutagenic frequency in Chinese hamster cells at the Na+/K+ ATPase locus was measured by resistance to ouabain following treatment with either photodynamic therapy (PDT) or UV irradiation. The C3H 10T 1/2 mouse embryo cell system was used to document the transformation frequency following PDT, UV irradiation, gamma irradiation or exposure to 3-methylcholanthrene (MCA). Treatments with UV irradiation were effective in producing mutants resistant to ouabain, and treatments with UV irradiation, gamma irradiation and MCA generated transformants at frequencies comparable to those which are reported in the literature. However, PDT treatment conditions (which produced a full range of cytotoxicity) did not induce any mutagenic or transformation activity above background levels.     

Heat sensitivity of Azotobacter vinelandii genetic transformation

Heating competent Azotobacter vinelandii at 37 or 42 degrees C resulted in a total loss of competence with no loss of viability. The transformation process was relatively insensitive to heating at either temperature once DNase-resistant DNA binding was nearly complete. Although competent and 42 degrees C-treated cells bound equivalent amounts of [32P]DNA in a DNase-resistant state, no donor DNA marker (nif) or radioactivity was detected in the envelope-free cell lysate of heated cells, suggesting that DNA transport across the cell envelope was a heat-sensitive event. Competence was reacquired in a 42 degrees C-treated culture after 2 h of incubation at 30 degrees C by a process which required RNA and protein syntheses. The release of a surface glycoprotein, required for competence, from cells treated at 42 degrees C occurred in an insufficient amount to account for the total loss of competence. Recovery of competence in 42 degrees C-treated cells and further transformation of competent cells were prevented by the exposure of cells to saturating amounts of transforming DNA. Further DNase-resistant DNA binding, however, still occurred, suggesting that there were two types of receptors for DNase-resistant DNA binding to competent A. vinelandii. DNase-resistant DNA binding was dependent on magnesium ions, and at least one receptor type did not discriminate against heterologous DNA.     

The DNA damage spectrum produced by simulated sunlight

The mutagenic effects of ultraviolet and solar irradiation are thought to be due to the formation of DNA photoproducts, most notably cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts ((6-4)PPs). Experimental systems for determining the levels and sequence dependence of photoproduct formation in DNA have often used high doses of short-wave (UVC) irradiation. We have re-assessed this issue by using DNA sequencing technologies and different doses of UVC as well as more physiologically relevant doses of solar irradiation emitted from a solar UV simulator. It has been questioned whether hot alkali treatment can detect (6-4)PPs at all sequence positions. With high UVC doses, the sequence distribution of (6-4)PPs was virtually identical when hot alkali or UV damage endonuclease (UVDE) were used for detection, which appears to validate both methods. The (6-4)PPs form at 5'-TpC and 5'CpC sequences but very low levels are seen at all other dipyrimidines including 5'-TpT. Contrary to expectation, we find that (6-4) photoproducts form at almost undetectable levels under conditions of irradiation for up to five hours with the solar UV simulator. The same treatment produces high levels of CPDs. In addition, DNA glycosylases, which recognize oxidized and ring-opened bases, did not produce significant cleavage of sunlight-irradiated DNA. From these data, we conclude that cyclobutane pyrimidine dimers are at least 20 to 40 times more frequent than any other DNA photoproduct when DNA or cells are irradiated with simulated sunlight.     

An unstable donor-recipient DNA complex in transformation of Bacillus subtilis.

Summary In re-extracted DNA obtained shortly after uptake of transforming DNA by Bacillus subtilis, increased amounts of donor DNA radioactivity banding at the position of donor-recipient DNA complex (DRC) are observed in CsCl gradients, if the cells are irradiated with high doses of UV prior to reextraction of the DNA. Qualitatively, the same phenomenon is observed if lysates of transforming cells are irradiated. UV-irradiation of lysates of competent cells to which single-stranded DNA is added after lysis, does not result in linkage of this DNA to the chromosomal DNA. Two observations argue in favour of the formation of a specific labile complex between donor and resident DNA during transformation. Firstly, heterologous donor DNA from Escherichia coli, although being processed to single-stranded DNA in competent B. subtilis, does not seem to be linked to the recipient chromosome upon UV-irradiation, and secondly, the labile complex of donor and recipient DNA can be stabilized by means of treatment of the lysates of transforming cells with 4, 5¹, 8-trimethylpsoralen in conjuction with long-wave ultra violet light irradiation. This indicates that base-pairing is involved in the formation of the complex. On the basis of these results we assume that the unstable complex of donor and recipient DNA is an early intermediate in genetic recombination during transformation.     

W-mutagenesis in competent cells of Bacillus subtilis

Summary The relative yield (N _m/N) of fluorescent mutants Ind^- after the transformation of Bacillus subtilis cells by means of UV-irradiated DNA is much higher in an uvr ^- recipient than in an uvr ⁺ strain, when compared at equal fluence, but practically identical at equal survival. Ind^- mutations are induced by UV-irradiation of separated single strands of transforming DNA. The H-strand is much more sensitive to the mutagenic action of UV light. Preliminary irradiation of competent recipient cells by moderate UV fluences increases the survival of UV-or -irradiated transforming DNA (W-reactivation) and the frequency of Ind^- mutations (W-mutagenesis). During transfection of B. subtilis cells by UV-irradiated prophage DNA isolated from lysogenic cells B. subtilis (Ø105 c ⁺) c-mutants of the phage are obtained in high yield only in conditions of W-mutagenesis, i.e. in UV-irradiated recipient cells. These data show that there is no substantial spontaneous induction of error-prone SOS-repair system in the competent cells of B. subtilis.     

Mutation induction with UV- and X-radiations in spores and vegetative cells of Bacillus subtilis.

Spores and vegetative cells of Bacillus subtilis strains with various defects in DNA-repair capacities (hcr-, ssp-, hcr-ssp-) were irradiated with UV radiation or X-rays. Induced mutation frequency was determined from the observed frequency of prototrophic reversion of a suppressible auxotrophic mutation. At equal physical dose, after either UV- or X-irradiation, spores were more resistant to mutations as well as to killing than were vegetative cells. However, quantitative comparison revealed that, at equally lethal doses, spores and vegetative cells were almost equally mutable by X-rays whereas spores were considerably less mutable by UV than were vegetative cells. Thus, as judged from their mutagenic efficiency relative to the lethality, X-ray-induced damage in the spore DNA and the vegetative DNA were equally mutagenic, while UV-induced DNA photoproducts in the spore were less mutagenic than those in vegetative cells. Post-treatment of UV-irradiated cells with caffeine decreased the survival and the induced mutation frequency for either spores or vegetative cells for all the strains. In X-irradiated spores, however, a similar suppressing effect of caffeine was observed only for mutability of a strain lacking DNA polymerase I activity.     

Mutation of Haemophilus influenzae transforming DNA in vitro with near-ultraviolet radiation: action spectrum

Mutations were produced in purified transforming DNA from Haemophilus influenzae by near-UV radiation and were assayed as mutants among cells transformed with irradiated DNA. The maximum efficiency of mutation induction was at around 334 nm, and the efficiency dropped off steeply at lower and higher wavelengths. The difference between the action spectrum for mutation and that for the oxygen-independent inactivation of transforming DNA, which had a shoulder at 365 nm, indicates that there are different lesions involved in the inactivating and mutagenic effects of near-UV. The presence of histidine during irradiation enhanced the mutagenic effect at 334 and 365 nm, although it protected against inactivation at 365 nm. The effective near-UV wavelengths for in vitro mutation are to some extent the same as the effective wavelengths for mutation in vivo reported previously. These findings indicate that mutations are produced in vivo by near-UV with DNA as the primary target molecule rather than by a secondary non-photochemical reaction between DNA and some other cell component.     

Induction of homologous recombination in Saccharomyces cerevisiae

Summary We have investigated the effects of UV irradiation of Saccharomyces cerevisiae in order to distinguish whether UV-induced recombination results from the induction of enzymes required for homologous recombination, of the production of substrate sites for recombination containing regions of DNA damage. We utilized split-dose experiments to investigate the induction of proteins required for survival, gene conversion, and mutation in a diploid strain of S. cerevisiae. We demonstrate that inducing doses of UV irradiation followed by a 6 h period of incubation render the cells resistant to challenge doses of UV irradiation. The effects of inducing and challenge doses of UV irradiation upon interchromosomal gene conversion and mutation are strictly additive. Using the yeast URA3 gene cloned in non-replicating single- and double-stranded plasmid vectors that integrate into chromosomal genes upon transformation, we show that UV irradiation of haploid yeast cells and homologous plasmid DNA sequences each stimulate homologous recombination approximately two-fold, and that these effects are additive. Non-specific DNA damage has little effect on the stimulation of, homologous recombination, as shown by studies in which UV-irradiated heterologous DNA was included in transformation/recombination experiments. We further demonstrate that the effect of competing single- and double-stranded heterologous DNA sequences differs in UV-irradiated and unirradiated cells, suggesting an induction of recombinational machinery in UV-irradiated S. cerevisiae cells.     

Respective roles of pyrimidine dimer and pyrimidine (6-4) pyrimidone photoproducts in UV mutagenesis of simian virus 40 DNA in mammalian cells.

UV light induces DNA lesions which are mutagenic in mammalian cells. We used simian virus 40 tsB201 (unable to produce viral capsid at the restrictive temperature of 41 degrees C because of a point mutation in the VP1 gene) to analyze the mutagenic potency of the two major UV-induced lesions, pyrimidine dimers (Py-Py) and pyrimidine (6-4) pyrimidones [Py(6-4)Py], which are formed on the same nucleotide sites. The mutagenesis criterion was the reversion toward a wild-type growth phenotype. After UV irradiation (mainly at 254 nm), part of the DNA was treated with the photoreactivating enzyme of Escherichia coli, which monomerizes Py-Py but does not modify the Py(6-4)Py photoproduct. Higher survival and lower mutation frequency rates for the photoreactivated DNA indicated that the two lesions were lethal and mutagenic. The VP1 gene of some mutants was entirely sequenced. The mutation spectra showed that the two lesions did not induce the same mutation hot spots, although some sites were common to both. The induced mutation hot spots were not only correlated with lesion hot spots but seemed partially directed by local DNA structures.     

Transformation of Saccharomyces cerevisiae with UV-irradiated single-stranded plasmid.

UV-irradiated single-stranded replicative plasmids were used to transform different yeast strains. The low doses of UV used in this study (10-75 J/m2) caused a significant decrease in the transforming efficiency of plasmid DNA in the Rad+ strain, while they had no effect on transformation with double-stranded plasmids of comparable size. Neither the rev3 mutation, nor the rad18 or rad52 mutations influenced the efficiency of transformation with irradiated single-stranded plasmid. However, it was found to be decreased in the double rev3 rad52 mutant. Extracellular irradiation of plasmid that contains both URA3 and LEU2 genes (psLU) gave rise to up to 5% Leu- transformants among selected Ura+ ones in the repair-proficient strain. Induction of Leu- transformants was dose-dependent and only partially depressed in the rev3 mutant. These results suggest that both mutagenic and recombinational repair processes operate on UV-damaged single-stranded DNA in yeast.     

Effect of transforming DNA on growth and frequency of mutation of Streptococcus pneumoniae.

We studied the effect of the presence of homologous transforming DNA on the growth of several transformable strains of Streptococcus pneumoniae and on the frequency of mutation of these strains to various antibiotic resistances. We observed no effect on growth until the strains became competent, when growth was depressed. At the end of the competence period, some strains showed recovery to varying degrees, whereas others showed evidence of cell death. Growth was also depressed by the presence of DNA from Escherichia coli, indicating that recombination was not likely to be the cause of the observed effect. Furthermore, cell death was not caused by the induction of a prophage. Several of the strains showed increased mutation frequencies during the competence period, although treatment with E. coli DNA gave no such effect, indicating that the mutagenesis was due to recombination. We observed no mutagenesis due to UV irradiation of the strains. The possibility that integration of the transforming DNA may produce lesions which induce error-prone repair is discussed. Furthermore, a strain that showed no mutability by transforming DNA, indicating the presence of a more efficient repair system, gave evidence of producing higher amounts of the hex system when competent, and the possible relationship between these properties is discussed.     

Test of models for the sequence specificity of UV-induced mutations in mammalian cells

We have used mathematical modeling and statistical analysis to examine the correlation between UV-induced DNA damage and resulting base-substitution mutations in mammalian cells. The frequency and site specificity of UV-induced photoproducts in the supF gene of the pZ189 shuttle vector plasmid were compared with the frequency and site specificity of base-substitution mutations induced upon passage of the UV-irradiated vector in monkey cells. The hypothesis that the observed mutational spectrum is due to a preferential insertion of adenosine opposite UV photoproducts in the DNA template was found to best explain the mutational data. Models in which it was postulated that only (6-4) photoproducts, and not cyclobutane dimers, are mutagenic, or that the relative frequency of photoproduct formation does not influence mutation frequencies, fit the data much less well. This analysis demonstrates that molecular mechanisms of mutagenesis in mammalian cells can be deduced from mutational data obtained with a shuttle vector system.     

The role of DNA polymerase eta in UV mutational spectra

UV irradiation generates predominantly cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts in DNA. CPDs are thought to be responsible for most of the UV-induced mutations. Thymine-thymine CPDs, and probably also CPDs containing cytosine, are replicated in vivo in a largely accurate manner by a DNA polymerase eta (Pol eta) dependent process. Pol eta is encoded by the POLH (XPV) gene in humans. In order to clarify the specific role of Pol eta in UV mutagenesis, we have used an siRNA knockdown approach in combination with a supF shuttle vector which replicates in mammalian cells. This strategy provides an advantage over studying mutagenesis in cell lines derived from normal individuals and XP-V patients, since the genetic background of the cells is identical. Synthetic RNA duplexes were used to inhibit Pol eta expression in 293T cells. The reduction of Pol eta mRNA and protein was greater than 90%. The supF shuttle vector was irradiated with UVC and replicated in 293T cells in presence of anti-Pol eta siRNA. The supF mutant frequency was increased by up to 3.6-fold in the siRNA knockdown cells relative to control cells confirming that Pol eta plays an important role in mutation avoidance and that the pol eta knockdown was efficient. UV-induced supF mutants were sequenced from siRNA-treated cells and controls. Surprisingly, neither the type of mutations nor their distribution along the supF gene were substantially different between controls and siRNA knockdown cells and were predominantly C to T and CC to TT transitions at dipyrimidine sites. The data are compatible with two models. (i) Incorrect replication of cytosine-containing photoproducts by a polymerase other than Pol eta produces similar mutations as when Pol eta is present but at a higher frequency. (ii) Due to lack of Pol eta or low levels of remaining Pol eta, lesion replication is delayed allowing more time for cytosine deamination within CPDs to occur. We provide proof of principle that siRNA technology can be used to dissect the in vivo roles of lesion bypass DNA polymerases in DNA damage-induced mutagenesis.     

Characterization of a conditionally transformation-deficient mutant of Haemophilus influenzae that carries a mutation in the rec-1 gene region.

A mutant of Haemophilus influenzae, designated HM5, carrying a mutation in the rec-1 gene region, is described. This mutant transformed approximately 100-fold less well than does the wild type, but approximately 100-fold better than rec1 mutants. The mutant was less sensitive to UV irradiation and less "reckless" than rec1 mutants. In contrast to rec1 lysogens, HP1c1 lysogens of the mutant were inducible, and during transformation, recombinant-type activity was formed to the same extent as in the wild type. Although the integration of donor DNA was complete, the integrated DNA was not replicated at 36 degrees C. Both the inhibition of replication of the donor-recipient DNA complex and the transformation deficiency could be suppressed when, after DNA entry, the cells were incubated under suboptimal conditions. The loss of colony formation after UV irradiation was suppressible by the same conditions.     

Mutator alleles of yeast DNA polymerase zeta

The yeast REV3 gene encodes the catalytic subunit of DNA polymerase zeta (pol zeta), a B family polymerase that performs mutagenic DNA synthesis in cells. To probe pol zeta mutagenic functions, we generated six mutator alleles of REV3 with amino acid replacements for Leu979, a highly conserved residue inferred to be at the pol zeta active site. Replacing Leu979 with Gly, Val, Asn, Lys, Met or Phe resulted in yeast strains with elevated UV-induced mutant frequencies. While four of these strains had reduced survival following UV irradiation, the rev3-L979F and rev3-L979M strains had normal survival, suggesting retention of pol zeta catalytic activity. UV mutagenesis in the rev3-L979F background was increased when photoproduct bypass by pol eta was eliminated by deletion of RAD30. The rev3-L979F mutation had little to no effect on mutagenesis in an ogg1Delta background, which cannot repair 8-oxo-guanine in DNA. UV-induced can1 mutants from rev3-L979F and rad30Deltarev3-L979F strains primarily contained base substitutions and complex mutations, suggesting error-prone bypass of UV photoproducts by L979F pol zeta. Spontaneous mutation rates in rev3-L979F and rev3-L979M strains are elevated by about two-fold overall and by two- to eight-fold for C to G transversions and complex mutations, both of which are known to be generated by wild-type pol zetain vitro. These results indicate that Rev3p-Leu979 replacements reduce the fidelity of DNA synthesis by yeast pol zetain vivo. In conjunction with earlier studies, the data establish that the conserved amino acid at the active site location occupied by Leu979 is critical for the fidelity of all four yeast B family polymerases. Reduced fidelity with retention of robust polymerase activity suggests that the homologous rev3-L979F allele may be useful for analyzing pol zeta functions in mammals, where REV3 deletion is lethal.