Oxidative damage in DNA. Lack of mutagenicity by thymine glycol lesions

Thymine glycol (5,6-dihydroxy-5,6-dihydrothymine) is a base damage common to oxidative mutagens and the major stable radiolysis product of thymine in DNA. We assessed the mutagenic potential of thymine glycols in single-stranded bacteriophage DNA during transfection of Escherichia coli wild-type and umuC strains. cis-Thymine glycols were induced in DNA by reaction with the chemical oxidant, osmium tetroxide (OsO4); modification of thymines was quantitated by using anti-thymine glycol antibody. Inactivation of transfecting molecules showed that one lethal hit corresponded to 1.5 to 2.1 thymine glycols per phage DNA in normal cells, whereas conditions of W-reactivation (SOS induction) reversed 60 to 80% of inactivating events. Forward mutations in the lacI and lacZ' (alpha) genes of f1 and M13 hybrid phage DNAs were induced in OsO4-treated DNA in a dose-dependent manner, in both wild-type and umuC cells. Sequence analysis of hybrid phage mutants revealed that mutations occurred preferentially at cytosine sites rather than thymine sites, indicating that thymine glycols were not the principal pre-mutagenic lesions in the single-stranded DNA. A mutagenic specificity for C----T transitions was confirmed by OsO4-induced reversion of mutant lac phage. Pathways for mutagenesis at derivatives of oxidized cytosine are discussed.     

Inducible reactivation of bacteriophage T7 damaged by methyl methanesulfonate or UV light.

We examined the effects of host mutations affecting "SOS"-mediated UV light reactivation on the survival of bacteriophage T7 damaged by UV light or methyl methanesulfonate (MMS). Survival of T7 alkylated with MMS was not affected by the presence of plasmid pKM101 or by a umuC mutation in the host. The survival of UV light-irradiated T7 was similar in umuC+ and umuC strains but was slightly enhanced by the presence of pKM101. When phage survival was determined on host cells preirradiated with a single inducing dose of UV light, these same strains permitted higher survival than that seen with noninduced cells for both UV light- and MMS-damaged phage. The extent of T7 reactivation was approximately proportional to the UV light inducing dose inflicted upon each bacterial strain and was dependent upon phage DNA damage. Enhanced survival of T7 after exposure to UV light or MMS was also observed after thermal induction of a dnaB mutant. Thus, lethal lesions introduced by UV light or MMS are apparently repaired more efficiently when host cells are induced for the SOS cascade, and this inducible reactivation of T7 is umuC+ independent.     

Effect of umuC mutations on targeted and untargeted ultraviolet mutagenesis in bacteriophage lambda

Mutagenesis of phage lambda towards clear-plaque phenotype (c+----c) results in two classes of mutants that can be distinguished genetically and morphologically. Indirect mutagenesis, i.e. mutagenesis of unirradiated phage lambda c+ stimulated by the ultraviolet irradiation of the Escherichia coli host, results in mixed bursts (c/c+) of turbid wild-type and clear-plaque mutant phages. Pure bursts of lambda c mutants are induced by irradiation of the phage genome. Irradiation of both phages and host bacteria stimulates the production of the two classes of mutant clones. We show that three different mutant alleles of the E. coli umuC gene only prevent the appearance of pure bursts of clear-plaque mutants, while mixed bursts are produced at least as frequently in umuC mutants as in the umuC+ parent.     

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.     

Non-targeted mutagenesis of unirradiated lambda phage in Escherichia coli host cells irradiated with ultraviolet light

Non-targeted mutagenesis of lambda phage by ultraviolet light is the increase over background mutagenesis when non-irradiated phage are grown in irradiated Escherichia coli host cells. Such mutagenesis is caused by different processes from targeted mutagenesis, in which mutations in irradiated phage are correlated with photoproducts in the phage DNA. Non-irradiated phage grown in heavily irradiated uvr+ host cells showed non-targeted mutations, which were 3/4 frameshifts, whereas targeted mutations were 2/3 transitions. For non-targeted mutagenesis in heavily irradiated host cells, there were one to two mutant phage per mutant burst. From this and the pathways of lambda DNA synthesis, it can be argued that non-targeted mutagenesis involves a loss of fidelity in semiconservative DNA replication. A series of experiments with various mutant host cells showed a major pathway of non-targeted mutagenesis by ultraviolet light, which acts in addition to "SOS induction" (where cleavage of the LexA repressor by RecA protease leads to din gene induction): (1) the induction of mutants has the same dependence on irradiation for wild-type and for umuC host cells; (2) a strain in which the SOS pathway is constitutively induced requires irradiation to the same level as wild-type cells in order to fully activate non-targeted mutagenesis; (3) non-targeted mutagenesis occurs to some extent in irradiated recA recB cells. In cells with very low levels of PolI, the induction of non-targeted mutagenesis by ultraviolet light is enhanced. We propose that the major pathway for non-targeted mutagenesis in irradiated host cells involves binding of the enzyme DNA polymerase I to damaged genomic DNA, and that the low polymerase activity leads to frameshift mutations during semiconservative DNA replication. The data suggest that this process will play a much smaller role in ultraviolet mutagenesis of the bacterial genome than it does in the mutagenesis of lambda phage.     

Alleviation of type I restriction in the presence of plasmids of group inc I. General characteristics and molecular cloning of the ard gene

The capability of a number of plasmids of incN and incI groups to alleviate an action of type I EcoK, EcoB, EcoD, and EcoA restriction endonucleases on the unmodified DNA was revealed. The efficiency of EcoK action on lambda 0 DNA is alleviated about 10 divided by 100 fold in E. coli K12 AB 1157 bacteria containing the plasmid of incN group (pKM101, N3, pJA4733) or incI group (R144, R648; R621a; ColIb-P9). We have cloned ard gene of ColIb-P9 plasmid (SalI-C fragment) in pBR322 multicopying vector. A hybrid clone abolishing the EcoK restriction has been received. Ard gene activity is independent of the recA, recBc, recF, lexA, umuC, lon bacterial genes activity. Ard gene's product does not inhibit the EcoK restriction endonuclease action as well as ocr protein (phage T7) and does not increase the process of methylation of DNA as well as ral protein of phage lambda.     

Analysis of the involvement of human N-acetyltransferase 1 in the genotoxic activation of bladder carcinogenic arylamines using a SOS/umu assay system

Human acetyltransferase genes NAT1 or NAT2 were expressed in a Salmonella typhimurium strain used to detect the genotoxicity of bladder carcinogens. To clarify whether the human and rodent bladder carcinogenic arylamines are activated via either NAT1 or NAT2 to cause genotoxicity, a SOS/umu genotoxicity assay was used, with the strains S. typhimurium NM6001 (NAT1-overexpressing strain), S. typhimurium NM6002 (NAT2-overexpressing strain), and S. typhimurium NM6000 (O-AT-deficient parent strain). Genotoxicity was measured by induction of SOS/umuC gene expression in the system, which contained both an umuC"lacZ fusion gene and NAT1 or NAT2 plasmids. 4-Aminobiphenyl, 2-acetylaminofluorene, beta-naphthylamine, o-tolidine, o-anisidine, and benzidine exhibited dose-dependent induction of the umuC gene in strain NM6001. Although the induction of umuC by these chemicals was observed in the NM6002 strain, the induction was considerably lower than in the NM6001 strain. In the parent strain, NM6000, none of these compounds induced umuC gene expression. We also determined activation of these chemicals by recombinant human cytochrome P450 (P450 or CYP) 1A2 enzyme in three S. typhimurium tester strains. The activation of the chemicals was stronger in the NM6001 strain than that in NM6002. The specific NAT1 inhibitor 5-iodosalicylic acid inhibited umuC gene expression induced by aromatic amines used. These results could provide evidence that the bladder carcinogenic aromatic amines are mainly activated by the NAT1 enzyme to produce DNA damage rather than NAT2. The NAT1-overexpressing strain can be used to determine the genotoxic activation of bladder carcinogenic arylamines in the umu test and could provide a tool for predicting the carcinogenic potential of arylamines.     

Construction of a highly error-prone DNA polymerase for developing organelle mutation systems

A novel family of DNA polymerases replicates organelle genomes in a wide distribution of taxa encompassing plants and protozoans. Making error-prone mutator versions of gamma DNA polymerases revolutionised our understanding of animal mitochondrial genomes but similar advances have not been made for the organelle DNA polymerases present in plant mitochondria and chloroplasts. We tested the fidelities of error prone tobacco organelle DNA polymerases using a novel positive selection method involving replication of the phage lambda cI repressor gene. Unlike gamma DNA polymerases, ablation of 3′–5′ exonuclease function resulted in a modest 5–8-fold error rate increase. Combining exonuclease deficiency with a polymerisation domain substitution raised the organelle DNA polymerase error rate by 140-fold relative to the wild type enzyme. This high error rate compares favourably with error-rates of mutator versions of animal gamma DNA polymerases. The error prone organelle DNA polymerase introduced mutations at multiple locations ranging from two to seven sites in half of the mutant cI genes studied. Single base substitutions predominated including frequent A:A (template: dNMP) mispairings. High error rate and semi-dominance to the wild type enzyme in vitro make the error prone organelle DNA polymerase suitable for elevating mutation rates in chloroplasts and mitochondria.     

Role of the umuC gene in postreplication repair in UV-irradiated Escherichia coli K-12 uvrB

The role of the umuC gene product in postreplication repair was studied in UV-irradiated Escherichia coli K-12 uvrB cells. A mutation at umuC increased the UV radiation sensitivities of uvrB, uvrB recF, uvrB recB, and uvrB recF recB cells; it also increased the deficiencies in the repair of DNA daughter-strand gaps in these strains, but it did not affect the repair of DNA double-strand breaks that arose from unrepaired DNA daughter-strand gaps. We suggest that the umuC gene product is involved in a minor system for the repair of DNA daughter-strand gaps, possibly the repair of overlapping DNA daughter-strand gaps.     

Mutational spectrum analysis of umuC-independent and umuC-dependent gamma-radiation mutagenesis in Escherichia coli

gamma-Radiation mutagenesis (oxic versus anoxic) was examined in wild-type, umuC and recA strains of Escherichia coli K-12. Mutagenesis [argE3(Oc)----Arg+] was blocked in a delta (recA-srlR)306 strain at the same doses that induced mutations in umuC122::Tn5 and wild-type strains, indicating that both umuC-independent and umuC-dependent mechanisms function within recA-dependent misrepair. Analyses of various suppressor and back mutations that result in argE3 and hisG4 ochre reversion and an analysis of trpE9777 (+1 frameshift) reversion were performed on umuC and wild-type cells irradiated in the presence and absence of oxygen. While the umuC strain showed the gamma-radiation induction of base substitution and frameshifts when irradiated in the absence of oxygen, the umuC mutation blocked all oxygen-dependent base-substitution mutagenesis, but not all oxygen-dependent frameshift mutagenesis. For anoxically irradiated cells, the yields of GC----AT [i.e., at the supB and supE (Oc) loci] and AT----GC transitions (i.e., at the argE3 and hisG4 loci) were essentially umuC independent, while the yields of (AT or GC)----TA transversions (i.e., at the supC, supL, supM, supN and supX loci) were heavily umuC dependent. These data suggest new concepts about the nature of the DNA lesions and the mutagenic mechanisms that lead to gamma-radiation mutagenesis.     

Weakening of bacteriophage lambda EcoK DNA restriction in the presence of plasmid pKM101 ard+. I. General characteristics and genetic localization

The host-controlled K-restriction of unmodified phage lambda is ten to hundred-fold alleviated in the E. coli K12 strain, carring plasmid pKM101 of N-incompatibility group. By restriction mapping Tn5 insertion in pKM101, which reduced pKM101-mediated alleviation of K-restriction, was shown to by located within BglII-B-fragment approximately 9 kb anticlockwise from the EcoRI-site of pKM101. We have termed the gene(s) promoting the alleviation of K-restriction ARD (Alleviation of Restriction of DNA). It was shown that (i) plasmid pKM101-mediated alleviation of K-restriction did not depend on bacterial genes LexA, RecBC, umuC and plasmid gene muc; (ii) ard gene did not mediate EcoK type modification of DNA and did not enhance the modification activity of EcoK system in a way similar to that observed with RAL gene of phage lambda. Action of Ard gene of plasmid pKM101 is highly specific: alleviation of restriction of DNA lambda takes place only in K-strains of E. coli and is practically absent in B-strains and also in E. coli strains which have restricting enzymes of 11 type, EcoRI and EcoRIII.     

Induction of SOS responses in Escherichia coli by 5-fluorouracil

The inducibility of SOS responses by 5-fluorouracil (5-FU), which has been used as an antitumor drug, was studied in Escherichia coli cells which have different DNA repair capacities for UV lesions. Expression of the umuC gene was apparently induced by 5-FU in the wild-type and uvrA strains, but not in lexA and recA strains. The inducibility of the umuC gene by 5-FU, the metabolite of which inhibits thymidylate synthetase, was abolished in cultures containing deoxythymidine monophosphate which is converted from deoxyuridine monophosphate by thymidylate synthetase. These results suggest that 5-FU may exert its SOS inducibility by inhibiting thymidylate synthetase and then disturbing DNA metabolism but not by incorporating 5-FU residues into RNA. Further, 5-FU weakly induced mutations in E. coli.     

Proteins required for ultraviolet light and chemical mutagenesis. Identification of the products of the umuC locus of Escherichia coli

umuC is the genetic locus showing the greatest specificity for the "error-prone repair" process that is responsible for u.v. and chemical mutagenesis in Escherichia coli. By generating a probe specific to umuC DNA, we have been able to clone the umuC locus. Through a combination of subcloning and Tn1000 mutagenesis, we have identified a region of 2.2 X 10(3) bases which contains the information necessary to complement umuC mutations. This region of DNA codes for two polypeptides with molecular weights of 16,000 and 45,000. The genes for these proteins are organized in an operon that is repressed by the lexA protein. Complementation of previously isolated umuC mutations revealed that these proteins correspond to two complementation groups, umuC, which codes for the 45,000 Mr protein, and umuD, which codes for the 16,000 Mr protein, and that therefore both proteins are essential for "error-prone repair" in E. coli.     

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.     

Action of plasmid ColIb-P9 on the survival after ultraviolet irradiation and on the mutagenesis of the imiC, uvm, recL, uvrE and tif1 sfiA lexA spr mutants of Escherichia coli K-12 cells

To clarify the mechanisms whereby the ColIb-P9 plasmid affects DNA repair processes, its effect was studied in mutant Escherichia coli K-12 cells with altered mutagenesis and DNA repair. The plasmid was shown to protect umuC, uvm, recL and uvrE mutants after UV irradiation. The frequency of UV-induced his+ revertants increased in the presence of the plasmid in umuC, uvm and recL mutant cells. The ColIb-P9 plasmid completely restored the UV mutability and survival of umuC mutants. These results suggest that the ColIb-P9 plasmid may encode a product similar to that of the umuC gene. In the tif1 sfiA lexA spr mutant cells where SOS functions are constitutively expressed, the ColIb-P9 plasmid increased the number of his+ revertants several times. This suggests that the action of ColIb-P9 is probably brought about not via the derepression of the recA gene but at the subsequent stages of the recA+lexA+-dependent DNA error-prone repair.     

Mechanism of SOS-induced targeted and untargeted mutagenesis in E. coli

This paper retraces the evolution of hypotheses concerning mechanisms of SOS induced mutagenesis. Moreover, it reports some recent data which support a new model for the mechanism of targeted and untargeted mutagenesis in E. coli. In summary, the SOS mutator effect, which is responsible for untargeted mutagenesis and perhaps for the misincorporation step in targeted mutagenesis, is believed to involve a fidelity function associated with DNA polymerase III and does not require the umuC gene product. umuC and umuD gene products are probably required specifically for elongation of DNA synthesis past blocking lesions, i.e. to allow mutagenic replication of damaged DNA.     

Inhibition of induction of adaptive response by o-vanillin in Escherichia coli B

Mutations induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were strongly enhanced in the presence of o-vanillin in E. coli B. The enhancement was also observed in uvrA, umuC, recA, polA, or alkB mutants. This effect was lower in an alkA mutant, but was restored in an alkA umuC double mutant. By contrast, the enhancing effect was almost blocked in an ada and ada umuC double mutant. It was necessary to add simultaneously MNNG and o-vanillin to the growth medium. Further investigations were conducted on the induction of ada and umuC genes using ada'-lacZ' and umuC'-lacZ' plasmids. o-Vanillin suppressed the induction of the ada gene by MNNG treatment, but not that of the umuC gene. In fact expression of the umuC gene was induced by lower concentrations of MNNG in the presence of o-vanillin. The results suggest that o-vanillin inhibits induction of the adaptive response, and consequently, the MNNG-induced mutation frequency is increased due to unrepaired O6-methylguanine.     

Y-family DNA polymerases in Escherichia coli

The observation that mutations in the Escherichia coli genes umuC+ and umuD+ abolish mutagenesis induced by UV light strongly supported the counterintuitive notion that such mutagenesis is an active rather than passive process. Genetic and biochemical studies have revealed that umuC+ and its homolog dinB+ encode novel DNA polymerases with the ability to catalyze synthesis past DNA lesions that otherwise stall replication--a process termed translesion synthesis (TLS). Similar polymerases have been identified in nearly all organisms, constituting a new enzyme superfamily. Although typically viewed as unfaithful copiers of DNA, recent studies suggest that certain TLS polymerases can perform proficient and moderately accurate bypass of particular types of DNA damage. Moreover, various cellular factors can modulate their activity and mutagenic potential.     

umuDC-mediated cold sensitivity is a manifestation of functions of the UmuD(2)C complex involved in a DNA damage checkpoint control

The umuDC genes are part of the Escherichia coli SOS response, and their expression is induced as a consequence of DNA damage. After induction, they help to promote cell survival via two temporally separate pathways. First, UmuD and UmuC together participate in a cell cycle checkpoint control; second, UmuD'(2)C enables translesion DNA replication over any remaining unrepaired or irreparable lesions in the DNA. Furthermore, elevated expression of the umuDC gene products leads to a cold-sensitive growth phenotype that correlates with a rapid inhibition of DNA synthesis. Here, using two mutant umuC alleles, one that encodes a UmuC derivative that lacks a detectable DNA polymerase activity (umuC104; D101N) and another that encodes a derivative that is unable to confer cold sensitivity but is proficient for SOS mutagenesis (umuC125; A39V), we show that umuDC-mediated cold sensitivity can be genetically separated from the role of UmuD'(2)C in SOS mutagenesis. Our genetic and biochemical characterizations of UmuC derivatives bearing nested deletions of C-terminal sequences indicate that umuDC-mediated cold sensitivity is not due solely to the single-stranded DNA binding activity of UmuC. Taken together, our analyses suggest that umuDC-mediated cold sensitivity is conferred by an activity of the UmuD(2)C complex and not by the separate actions of the UmuD and UmuC proteins. Finally, we present evidence for structural differences between UmuD and UmuD' in solution, consistent with the notion that these differences are important for the temporal regulation of the two separate physiological roles of the umuDC gene products.