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 photoreactivation on mutagenesis of lambda phage by ultraviolet light

There is disagreement in the literature as to whether the major mutagenic photoproduct induced in DNA by ultraviolet light is the cyclobutane dipyrimidine dimer, the most common product, or the [6-4] photoproduct, the next most frequent. In the experiments reported here, cyclobutane dimers were removed from irradiated lambda phage DNA by enzymatic photoreactivation, a process thought to affect no other photoproduct. Photoreactivation of lambda phage in host cells and of lambda DNA in solution reduced clear plaque mutants per plaque-forming unit by two-thirds, in host cells with a constant and near-maximal expression of the SOS functions required for mutagenesis. This result is interpreted to mean that removal of cyclobutane dimers in or near the mutated gene reduces mutation induced by ultraviolet light by two-thirds; therefore, cyclobutane dimers in the phage DNA are responsible for most observed mutations. DNA sequences of mutations in photoreactivated phage showed a smaller fraction of G.C to A.T transitions and a larger fraction of A.T to G.C transitions, compared to phage that were not photoreactivated. This suggests that cyclobutane dimers at TC and CC sites are particularly mutagenic.     

Induced reactivity of UV-damaged phage gamma in E. coli K12 host cells treated with aflatoxin B1 metabolites.

The metabolites of aflatoxin B1, the most potent hepatocarcinogen so far known, promote in E. coli K12 cells the reactivation of phage lambda damaged by ultraviolet (UV) radiation. This reactivation process is error prone; 25% of the phage DNA lesions are repaired, but mutagenesis, scored as clear plaque formation, is increased as much as 10-fold. Such reactivation of UV-damaged phage lambda, which occurs in wild-type and in uvrA but not in recA bacteria, is inducible: phage reactivation is obtained even after a long delay following treatment of the host by the short-lived metabolites. This induced reactivation of UV-damaged phage in hosts treated with metabolites of aflatoxin B1 is similar to direct of indirect UV reactivation. Metabolites of aflatoxin B1 produce induced phage reactivation as well as prophage lambda induction in lysogens and cell filamentation in non-lysogens. These cellular events are also triggered by DNA lesions caused by UV radiation and result from the induction of a metabolic pathway (SOS functions). We postulate that, in eucaryotes, carcinogens may induce cellular SOS functions similar to those in E. coli. Induction of such functions might be responsible for the transformation of mammalian cells.     

Kinetics of induction of error-prone repair of bacteriophage lambda by temperature shift in an Escherichia coli dnaB mutant.

Summary Preincubation at 42^o, before infection at permissive temperature by phage , of an Escherichia coli dnaB mutant, provokes a significant increase in survival and mutagenesis of ultraviolet irradiated phage as well as mutagenesis of untreated phage. Similarly to UV irradiation and many chemical mutagens, the inhibition of DNA synthesis by temperature shift of this dnaB mutant induces SOS repair. This work shows that replication blockage in bacterial DNA is not only mutagenic for bacterial DNA itself (Witkin, 1975) but also for normally replicating DNA, probably due to induction of diffusible products.     

Changes in DNA Base Sequence Induced by Gamma-Ray Mutagenesis of Lambda Phage and Prophage

Mutations in the cI (repressor) gene were induced by gamma-ray irradiation of lambda phage and of prophage, and 121 mutations were sequenced. Two-thirds of the mutations in irradiated phage assayed in recA host cells (no induction of the SOS response) were G:C to A:T transitions; it is hypothesized that these may arise during DNA replication from adenine mispairing with a cytosine product deaminated by irradiation. For irradiated phage assayed in host cells in which the SOS response had been induced, 85% of the mutations were base substitutions, and in 40 of the 41 base changes, a preexisting base pair had been replaced by an A:T pair; these might come from damaged bases acting as AP (apurinic or apyrimidinic) sites. The remaining mutations were 1 and 2 base deletions. In irradiated prophage, base change mutations involved the substitution of both A:T and of G:C pairs for the preexisting pairs; the substitution of G:C pairs shows that some base substitution mechanism acts on the cell genome but not on the phage. In the irradiated prophage, frameshifts and a significant number of gross rearrangements were also found.     

Damaged-site independent mutagenesis of phage lambda produced by inducible error-prone repair

The existence of damaged-site independent mutagenesis is confirmed here by scoring the appearance of clear-plaque (c-) or virulent (vir) forward mutations on intact (non-irradiated) phage lambda grown on UV-irradiated E. coli K12 hosts. The mutation frequency was measured as a function of the incubation time between the occurrence of host DNA lesions and phage infection. The time course of mutagenesis of intact phage followed the induction pattern observed upon UV-reactivation of UV-damaged phage by Defais et al. (1976). Intact phage did not mutate in UV-irradiated hosts carrying the uvm-25 mutation known to prevent the occurrence of UV-reactivation. These findings suggest that damaged-site independent mutagenesis results from inducible error-prone repair. Clear-plaque mutations arising on intact phage were mostly found in phage bursts consisting of clear and turbid plaque formers whereas UV-damaged phage gave rise to mostly clear-plaque formers. Contrarily to damaged-site dependent mutagenesis, damaged-site independent mutagenesis can arise even at late times during the phage replication cycle. Our data indicate that about half of the phage mutations that arise upon UV-reactivation are damaged-site independent mutations. Replication of intact phage DNA in a host during induction of SOS functions provides a sensitive assay for the detection of damaged-site independent mutagenesis.     

Conditionally lethal amber mutations in the dnaA region of the Escherichia coli chromosome that affect chromosome replication.

Three amber mutations, dna-801, dna-803, and dna-806, were isolated by localized mutagenesis of the dnaA-oriC region of the chromosome from an Escherichia coli strain carrying temperature-sensitive amber suppressors. When the mutations were not suppressed at 42 degrees C, the cells did not grow and DNA synthesis was arrested. They were very closely linked to each other and to the dnaA46 mutation. The mutant phenotype of each strain was converted to the wild type by infecting the mutants with specialized transducing phase lambda i21 dnaA-2 but not with lambda i21 tna. Derivatives of lambda i21 dnaA-2, each of which carried the amber mutation dna-801 dna-803, or dna-806, converted the dnaA mutant phenotype to Dna+ but did not convert rhe amber mutants to the wild-type phenotype. E. coli uvrB cells were irradiated with ultraviolet light and infected with each of these phage strains. An analysis of proteins synthesized in the cells revealed that two proteins with molecular weights of 50,000 and 43,000 were specified by lambda i21 dnaA-2 but not by lambda i21 tna. When the ultraviolet-irradiated cells did not carry an amber suppressor, the derivative phage with the amber mutation invariably failed to produce the 43,000-dalton protein, but when the host cell carried supF (tyrT), the protein was produced. The 50,000-dalton protein was unaffected.     

Different mechanisms for SOS induced alleviation of DNA restriction in Escherichia coli.

The alleviation of DNA restriction during the SOS response in Escherichia coli has been further investigated. With the EcoK DNA restriction system UV irradiated wild-type cells show a 10(4)-fold increase in ability to plate non-modified lambda phage and a 3-4 fold increase in transformation by non-modified plasmid DNA. A role for the umuDC genes of E coli in the process of SOS-induced restriction alleviation was identified by showing that a umuC122::Tn5 mutant could alleviate EcoK restriction to only 5% that of wild-type levels. Although umuDC are better characterized for their pivotal role in SOS induced mutagenesis, it is demonstrated here that umu-dependent alleviation of EcoK restriction is a transient process in which umu-dependent mutagenesis plays little part. A second form of SOS induced alleviation of DNA restriction is described in this paper involving the McrA restriction system. The mcrA gene is shown to be encoded within a defective prophage called e14 situated at the 25 min region on the Escherichia coli genetic map. e14 is known to abortively excise from the chromosome after SOS induction and it is demonstrated in this report that mcrA is lost from the genome after SOS induction as part of e14. This results in co-ordinate decrease in the level of McrA restriction within a population of cells.     

SOS mutator effect in E. coli mutants deficient in mismatch correction.

We have used bacteriophage lambda to characterize the mutator effect of the SOS response induced by u.v. irradiation of Escherichia coli. Mutagenesis of unirradiated phages grown in irradiated or unirradiated bacteria was detected by measuring forward mutagenesis in the immunity genes or reversion mutagenesis of an amber codon in the R gene. Relative to the wild-type, the SOS mutator effect was higher in E. coli mismatch correction-deficient mutants (mutH, mutL and mutS) and lower in an adenine methylation-deficient mutant ( dam3 ). We conclude that a large proportion of SOS-induced 'untargeted' mutations are removed by the methyl-directed mismatch correction system, which acts on newly synthesized DNA strands. The lower SOS mutator effect observed in E. coli dam mutants may be due to a selective killing of mismatch-bearing chromosomes resulting from undirected mismatch repair. The SOS mutator effect on undamaged lambda DNA, induced by u.v. irradiation of the host, appears to result from decreased fidelity of DNA synthesis.     

Mutagenesis of lambda phage: Weigle mutagenesis is induced by coincident lesions in the double helical DNA of the host cell genome

Summary We have studied the increase in mutation in mutagenized lambda phage when the host cells are also irradiated with ultraviolet light, Weigle mutagenesis. The increase in mutation is induced mainly by coincidences between a radiation-produced lesion in one strand of the host cell DNA and a second lesion in the complementary strand. This conclusion is based on experiments in which incorporation of the base analog bromouracil sensitized the host cells to ultraviolet light. For the same number of bromouracils incorporated per cell, uniform substitution gave a higher level of Weigle mutagenesis than did substitution in only one strand of the DNA double helix. The data also show some induction of Weigle mutagenesis by processes linear in ultraviolet fluence; possibilities include: lesions involving both complementary strands such as crosslinks, lesions in one strand opposite pre-existing discontinuities in the complementary strand, and very small contributions to induction from lesions in one strand only of the DNA.     

Suppression of tif-mediated induction of SOS functions in Escherichia coli by an altered dnaB protein.

The tif-1 mutation in the Escherichia coli recA gene is known to cause induction of the various "SOS" functions at high temperature, including massive synthesis of the recA protein, lethal filamentation, elevated mutagenesis, and, in lambda lysogens, induction of prophage. It is shown here that the deoxyribonucleic acid initiation mutation dnaB252 suppresses all these manifestations of tif expression. Induction of lambda by ultraviolet irradiation, however, is not affected by the dnaB252 mutation. No similar suppression of tif is observed with other dnaB mutations affecting deoxyribonucleic acid elongation or with other deoxyribonucleic acid initiation mutations at the dnaA and dnaC loci. The fact that an alteration of the dnaB protein specifically suppresses tif-mediated SOS induction implies a role of the replication apparatus in this process, as has been suggested for ultraviolet induction. The induction of lambda is known to proceed via repressor cleavage, presumably promoted by an activated (protease) form of the recA protein. Since lambda induction is normal after ultraviolet irradiation of the tif-1 dnaB252(lambda) strain, tif-mediated induction in this strain may be blocked in a tif-specific step leading to activation of the recA (tif) protein. It is possible that the recA (tif) mutant protein may be directly involved in the replication complex in processes leading to this activation.     

Role of the E. coli umuC gene product in the repair of single-stranded DNA phage

The umuC product of Escherichia coli has been suggested to have a central role in SOS induced error prone replication of DNA (Kato and Shinoura 1977). To investigate this possibility, we examined the effect of umuC mutations on error prone repair of single and double-stranded DNA phages. No Weigle reactivation of M13 phage was detected in a umuC mutant. Reactivation of lambda phage was reduced but still evident. However mutagenesis occurred in both cases. These results suggest that induced error prone replication of phage DNA can occur via umuC dependent (transdimer synthesis) and umuC independent mechanisms.     

Mutagenesis of ultraviolet-irradiated lambda phage by host cell irradiation: induction of Weigle mutagenesis is not an all-or-none process

Summary Ultraviolet mutagenesis of lambda phage to clear plaque formers is the same in the total phage population and in subpopulations of phage which have also mutated to gam ^- or at an amber codon. This is true for phage assayed in host cells in which Weigle mutagenesis has been either partially induced by low levels of ultraviolet irradiation, or fully induced by higher levels. If induction of Weigle mutagenesis were all-or-none, clear plaque formers in phage subpopulations selected for another mutation elsewhere would come mainly from induced cells; then the clear plaque mutation rate would always be that for fully induced host cells. Therefore, induction requires more than one lesion in host cell DNA.Although thymine starvation of cells induces synthesis of recA protein, it does not induce Weigle mutagenesis; in fact starvation inhibits induction of this process on subsequent ultraviolet irradiation of the cells.     

Ultraviolet light-induced mutagenesis in the Escherichia coli chromosome. Sequences of mutants in the cI gene of a lambda lysogen

DNA sequences were determined for 56 mutations induced by ultraviolet light in the lambda cI gene of an Escherichia coli uvr+ lysogen, which should reflect those occurring in the E. coli chromosome. The spectrum of mutagenesis was similar to that found in the cI gene of irradiated phase assayed in uvr- host cells, except that the fraction of transversions is about 35% in prophage and about 15% in phage. The cause of this difference is not known. Of 17 frameshifts in phage and prophage, six have an accompanying base substitution. These double mutational events are consistent with a model in which a photoproduct in a template can cause a DNA polymerase to insert a wrong base and destabilize the next few bases added, thus leading to a frameshift by a slippage mechanism.     

Separation of the SOS-dependent and SOS-independent components of alkylating-agent mutagenesis.

Escherichia coli plasmids containing the rpsL+ gene (Strs phenotype) as the target for mutation were treated in vitro with N-methyl-N-nitrosourea. Following fixation of mutations in E. coli MM294A cells (recA+ Strs), an unselected population of mutant and wild-type plasmids was isolated and transferred into a second host, E. coli 6451 (recA Strr). Strains carrying plasmid-encoded forward mutations were then selected as Strr isolates, while rpsL+ plasmids conferred the dominant Strs phenotype in the second host. Mutation induction and reduced survival of N-methyl-N-nitrosourea-treated plasmids were shown to be dose dependent. Because this system permitted analysis and manipulation of the levels of certain methylated bases produced in vitro by N-methyl-N-nitrosourea, it afforded the opportunity to assess directly the relative roles of these bases and of SOS functions in mutagenesis. The methylated plasmid DNA gave a mutation frequency of 6 X 10(-5) (a 40-fold increase over background) in physiologically normal cells. When the same methylated plasmid was repaired in vitro by using purified O6-methylguanine DNA methyltransferase (to correct O6-methylguanine and O4-methylthymine), no mutations were detected above background levels. In contrast, when the methylated plasmid DNA was introduced into host cells induced by UV light for the SOS functions, rpsL mutagenesis was enhanced eightfold over the level seen without SOS induction. This enhancement of mutagenesis by SOS was unaffected by prior treatment of the DNA with O6-methylguanine DNA methyltransferase. These results demonstrate a predominant mutagenic role for alkylation lesions other than O6-methylguanine or O4-methylthymine when SOS functions are induced. The mutation spectrum of N-methyl-N-nitrosourea under conditions of induced SOS functions revealed a majority of mutagenic events at A . T base pairs.     

Timing of ultraviolet light induced mutagenesis in simian virus 40 relative to viral DNA replication in monkey cells

Summary Simian virus 40 (SV40) was used to probe ultraviolet light (UV) — induced mutation in mammalian cells. Viral mutations were scored as reversions of early and late temperature-sensitive (ts) mutants to the wild-type (WT) phenotype. When virus was exposed to moderate or high UV doses, WT revertants were obtained at a frequency related to the square of the dose from two early (tsA) and one late (tsBC) mutant grown at the restrictive temperature. The reversions generated in the progeny of UV-irradiated early mutants presumably arose before the onset of viral DNA replication because, at the non-permissive temperature, tsA mutants are unable to express the functions responsible for the initiation of viral DNA synthesis. Moreover, the early mutant tsA209 underwent similar levels of induced reversion at the permissive and restrictive temperatures, suggesting that the pre-replicative mutational pathway might predominate for moderately and heavily irradiated virus, even under conditions where DNA synthesis can be initiated. The analysis of bursts from revertant plaques produced at the restrictive temperature was consistent with this interpretation. Although the mechanism of pre-replicative mutagenesis is not known, it is likely to be mediated by cellular activities owing to the low genetic complexity of the virus.     

Para-aminobenzoic acid inhibits a set of SOS functions in Escherichia coli K12

PABA - Vitamin H1 of group B, has obtained increasing fundamental interest as a very potent natural antimutagen after a series of our publications since 1979. In the first set of our experiments, we studied PABA in the assays with the alkylating agent N-methyl-N-nitrosourea (MNU). Mutagenic efficiency of this agent was suppressed up to 10-fold when PABA was administered into Escherichia coli cells concurrently with the mutagen or prior to the mutagenic treatment. NMR spectrometric and UV-spectrophotometric measurements did not reveal an interaction between the direct acting MNU and PABA, typical for some N-nitroso compounds and phenolics. PABA suppressed the error-prone DNA repair pathway induced by UV-irradiation. PABA decreased MNU-induced phage lambda lysogenic induction more than two orders of magnitude. PABA inhibited the thermal shift up to 400-fold in phage lambda from the permissive to non-permissive temperature in E. coli mutant tif-1 and decreased about two-fold W-reactivation of UV-damaged phage lambda. Chloramphenicol treatment of the cells just after the mutagenic treatment prevented the occurrence of PABA specific activity. The results suggest that PABA affects the SOS DNA repair pathway and the mutagenic response of E. coli. PABA appears to be an effective bioantimutagen reducing mutagenesis by modulating the error-prone DNA repair (SOS) response.     

Survival and mutagenesis of bacteriophage T7 damaged by methyl methanesulfonate and ethyl methanesulfonate

We have examined survival and mutagenesis of bacteriophage T7 after exposure to the alkylating agents methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS). It was found that although both alkylating agents caused increased reversion of specific T7 mutations, EMS caused a higher frequency of reversion than did MMS. Exposure of the host cells to ultraviolet light so as to induce the SOS system resulted in increased survival (Weigle reactivation) of T7 phage damaged with either EMS or MMS. However, after SOS induction of the host we did not detect an accompanying increase in mutation frequency measured as either reversion of specific T7 mutants or by generation of mutations in the T7 gene that codes for phage ligase. Neither mutation frequency nor survival of alkylated phage was affected by the umuD,C mutation in the Escherichia coli host nor by the presence of plasmid pKM101. This may mean that the mode of Weigle reactivation that is detected in T7 is not mutagenic in nature.     

Expression of ultraviolet-induced restriction alleviation in Escherichia coli K-12. Detection of a lambda phage fraction with a retarded mode of DNA injection

Ultraviolet-induced restriction alleviation is an SOS function which partially relieves the K-12-specific DNA restriction in Escherichia coli. Restriction alleviation is determined by observing elevated survival of unmodified phage lambda in cells irradiated with ultraviolet prior to infection. We demonstrate that restriction of lambda is also relieved when log-phase cells are irradiated as late as 50 min after adsorption of lambda. At this time more than 60% of the lambda DNA is already released as acid-soluble material from the cells. Experiments involving reextraction of lambda DNA from infected cells and a mild detergent treatment removing absorbed phages from the cellular surface showed that only a small specific fraction of all lambda infections is destined to escape restriction due to restriction alleviation. This fraction (10-20%) has a retarded mode of DNA injection (60 min or longer) after adsorption which allows the expression of the restriction alleviation function before the phage DNA is exposed to restriction endonucleases. This behaviour of a fraction of lambda phages explains why the SOS function restriction alleviation could initially be discovered. We show that the retarded mode of DNA injection is not required for another SOS function acting on lambda DNA, the increased repair of ultraviolet-irradiated DNA (Weigle reactivation).     

W-mutagenesis in the bisulfite-treated lambda phage

Survival of phage lambda cI857 inactivated by bisulfite (pH 5.6, 37 degrees C) is higher (the dose modification factor approx. 1.2) and frequency of bisulfite-induced c-mutations 2-4-fold lower on the lawn of the wild-type strain ung+, as compared to ung-1 mutant deficient in uracil-DNA glycosylase. Irradiation of host cells by a moderate UV dose inducing SOS repair system enhances the frequency of bisulfite-induced c-mutations 2-3-fold in the wild-type (ung+) host, but not in the ung-1 mutant. It is suggested that W-mutagenesis in bisulfite-treated lambda phage in the ung+ cells is due to SOS repair of apyrimidinic sites which are produced during excision of uracil residues, the products of cytosine deamination.