UV mutagenesis in E. coli with excision repair initiated by uvrABC or denV gene products

Mutation frequency responses produced by ultraviolet light are compared in 4 closely related strains of E. coli B/r having the same tyr(Oc) allele and different excision-repair capabilities: uvr+ (excision repair initiated by wild-type UvrABC activity), uvrA (excision repair defective), uvrA/pdenV-7 (excision repair initiated by endonuclease V of bacteriophage T4, DenV activity), and uvr+/pdenV-7 (excision repair initiated by UvrABC and DenV activities). The production of Tyr+ prototrophic mutants is classified into back-mutations and de novo or converted glutamine tRNA suppressor mutations to indicate different mutation events. Cells transformed with the plasmid pdenV-7 require larger exposures than the parent strains to produce comparable mutation frequency responses, indicating that DenV activity can repair mutagenic photoproducts. When damage reduction by UvrABC or DenV is compared for each of the specific categories of mutation, the results are consistent with the idea that pyrimidine dimers infrequently or never target back-mutations of this allele, frequently target the de novo suppressor mutations, and extensively or exclusively target the converted suppressor mutations. This analysis is based on the distinction that UvrABC-initiated excision repair recognizes dimer and non-dimer (pyrimidine (6-4) pyrimidone) photoproducts but that DenV-initiated repair recognizes only pyrimidine dimers.     

Mutagenic DNA repair in Escherichia coli. XV. Mutation frequency decline of ochre suppressor mutations in umuC and lexA bacteria occurring between ultraviolet irradiation and delayed photoreversal

Tryptophan-independent mutations were induced in CM1141 trpE65 umuC122::Tn5 following exposure to ultraviolet light (UV) plus delayed photoreversal. The mutations appeared to be exclusively class 2 ochre suppressors and showed mutation frequency decline (MFD) when the bacteria were incubated in glucose-salts medium after UV and before photoreversal. The phenomenon was similar to MFD after normal UV mutagenesis of umu+ bacteria, being inhibited in the presence of caffeine or in the absence of glucose. Mutations were also induced by UV plus delayed photoreversal in the lexA (Ind-) strain CM561, and the yield was higher than in the umuC strain suggesting that potentially mutagenic configurations might be removed or altered to some extent by the product of a gene under lexA control such that fewer were available for misincorporation events. MFD was also demonstrated in CM561 showing that this process is not dependent on the derepression of any genes under lexA control. MFD could still be demonstrated 23 min after UV at a time when most misincorporations seem to have occurred, but for technical reasons the possibility could not be excluded that the misincorporations in question could have occurred during rather than before the exposure to photoreversing light. Delayed photoreversal mutagenesis of normally non-UV-mutable strains has been interpreted as a first stage (misincorporation) of normal UV mutagenesis. The present results are consistent with this interpretation since MFD of suppressor mutations is a feature of both processes.     

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.     

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.     

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.     

Mutation probe of gene structure in E. coli: suppressor mutations in the seven-tRNA operon

Summary Cells defective in uracil-DNA glycosylase (ung:: Tn10) were used in two ways to reveal differences in select point mutations (GC to AT transitions) within the seven-tRNA operon of E. coli. The mutations were indicated as de novo or converted glutamine tRNA suppressor mutations in the genes glnU and/or glnV: (1) the kinetics of photoenzymatic monomerization of pyrimidine dimers quantitated by ung-dependent UV mutagenesis indicated more rapid repair of dimers at sites for converted suppressor mutation than of dimers at sites for de novo suppressor mutation, and (2) spontaneous deamination of cytosine was considerably more frequent at sites for converted suppressor mutation than at sites for de novo suppressor mutation. To explain these results we suggest the physical structure of the DNA in vivo is different at different sites in the seven-tRNA operon. The non-transcribed strand including specifically the anticodon region of the site for converted suppressor mutation may frequently be looped out in a single strand so that a T=C dimer is more accessible to DNA photolyase or a free cytosine residue of non-irradiated DNA is in an aqueous environment conducive to deamination. In addition, we analysed the spontaneous de novo suppressor mutation data to determine an estimate for the in vivo rate of cytosine deamination in double strand DNA of 3.2×10¹³/sec.     

Mutagenic DNA repair in Escherichia coli. XIII. Proofreading exonuclease of DNA polymerase III holoenzyme is not operational during UV mutagenesis

We have introduced a mutD5 mutation (which results in defective 3'-5'-exonuclease activity of the epsilon proofreading subunit of DNA polymerase III holoenzyme) into excision-defective Escherichia coli strains with varying SOS responses to UV light. MutD5 increased the spontaneous mutation frequency in all strains tested, including recA430, umuC122::Tn5, and umuC36 derivatives. It had no effect on UV mutability or immutability in any strain or on misincorporation revealed by delayed photoreversal in UV-irradiated umuC36, umuC122::Tn5, or recA430 bacteria. It is concluded that the epsilon proofreading subunit of DNA polymerase III holoenzyme is excluded, inhibited, or inoperative during misincorporation and mutagenesis after UV.     

Diverse backmutations at an ochre defect in the tyrA gene sequence of E. coli B/r

A DNA fragment including most of the tyrA gene from E. coli B/r strain WU (Tyr-, Leu-) was amplified in vitro by polymerase chain reaction. The sequence was determined, first, for essentially all of the fragment to locate an ochre nonsense defect, and second, repeatedly for a region of the fragment from several independent isolates containing backmutations at the ochre codon (spontaneous and UV-induced). There were 20 single base differences in the tyrA gene region from the analogous wild-type E. coli K12 sequence: an ochre codon at amino acid position 161, 18 silent changes (1 at the first codon base and 17 at the third) and one replacement of valine by alanine. Different backmutations at the ochre codon encoded lysine, glutamine, glutamic acid, leucine, cysteine, phenylalanine, serine or tyrosine. The diversities of base substitutions at the ochre codon after UV mutagenesis or after mutagenesis where targeting by dimers was reduced or eliminated (after photoreversal of irradiated cells treated with nalidixic acid to induce SOS functions or after UV mutagenesis of cells containing amplified DNA photolyase) were similar (with two notable exceptions). The overall differences between the gene sequences for E. coli K12 or B/r seemed consistent with the neutral theory of molecular evolution.     

Mutagenic DNA repair in Escherichia coli. XIX. On the roles of RecA protein in ultraviolet light mutagenesis

An experimental system was used in which His+ mutations induced by ultraviolet light (UV) arise from non-photo-reversible photoproducts whereas lethality is largely determined by photoreversible photoproducts. By exposing a strain with a deletion through recA to light immediately after UV, it was possible to examine mutagenesis under conditions where survival was not significantly different from 100%. No UV mutagenesis was seen in the absence of RecA protein even though the rest of the SOS system was fully expressed due to the presence of a defective LexA repressor and the active carboxy-terminal fragment of UmuD was present as a result of an engineered plasmid-borne gene. We conclude that RecA protein has a third essential function if UV mutagenesis is to be detected in excision-deficient-bacteria. Another experiment showed that in exerting this function RecA protein does not need activation by pyrimidine dimers elsewhere on the genome, in contrast to its protein-cleavage mediation functions with LexA and UmuD proteins. RecA1730 protein blocked UV mutagenesis unless delayed photoreversal was given showing that the third function of RecA protein is not in the misincorporation step. It is therefore most likely to be in the bypass step where UmuD' and UmuC are postulated to act, although the possibility cannot be excluded that RecA protein is required for some other survival function distinct from translesion synthesis.     

DNA photolyase in E. coli: effects on UV mutagenesis by plasmids expressing the phr gene

Derivatives of an E. coli plasmid pKY33 are described having specific insertions or deletions that effect or do not effect the phr gene (for DNA photolyase) carried in this plasmid. The various plasmids are tested to determine which cause an inhibition of UV mutagenesis producing glutamine tRNA ochre suppressor mutations. The inhibition is found to require a functional phr gene, which substantiates our earlier report that amplified DNA photolyase interferes specifically with a category of mutagenesis involving targeting by a pyrimidine dimer.     

In vivo deamination of cytosine-containing cyclobutane pyrimidine dimers in E. coli: a feasible part of UV-mutagenesis

We have estimated in vivo deamination rates for cytosines in cyclobutane pyrimidine dimers (CPD or PyPy) in UV-irradiated E. coli deficient in uracil DNA glycosylase. The protocol consisted of UV-irradiation, holding in buffer to allow for deamination of cytosines in CPDs and photoreversal (PR) to establish uracils where cytosines in CPD deaminated. The deamination rate at TC photoproducts targeting glutamine tRNA suppressor mutations was estimated from the increase in the mutation frequency after PR (MF(PR)) that developed as UV-irradiated cells were held before PR. Evidence suggested that an earlier study with this protocol under-estimated the deamination rate at sites producing the same mutations in an E. coli B/r strain. With a K12 strain, where the targeting apparently is principally by CPD and not (6-4) photoproducts, a larger rate of k = 0.0091 min(-1) at 42 degrees C resulted. The dark assay for MF also increased significantly with time for deamination consistent with a model for efficient mutation by translesion synthesis at uracil-containing CPD. In addition, we used a strain constructed by Cupples and Miller in which beta-galactosidase was inactive because -GGG- was at codon 461 and would revert to Lac(+) only when replaced by -GAG- or -GAA- for glutamate. CC photoproducts at this target site in the opposite DNA strand could reveal effects of first and second deaminations in the same CPD. MF(PR) for Lac(+) mutations increased and then decreased as a function of deamination time (at six temperatures 36-48 degrees C). Fitting an approximate model equation that distinguished two different deamination rates to these data suggested a first deamination producing Lac(+) at a rate about eight-fold less than a second deamination restoring the Lac(-) phenotype. We conclude that deamination, changing a cytosine-containing CPD to a uracil-containing CPD, could be an integral part of UV-induced C-to-T mutations.     

Anti-mutagenic effect of ultraviolet light on spontaneous tyrosine tRNA ochre suppressor mutations in Escherichia coli

Summary The numbers of tyrosine tRNA ochre suppressor mutations arising spontaneously or after UV irradiation in different strains of Escherichia coli K12 are considered. The DNA sequence change requisite for this type of mutation would be a transversion at a cytosine between two purines, where pyrimidine-pyrimidine photoproducts could not form. We find that UV mutagenesis does not produce these tyrosine tRNA ochre suppressor mutations. With lexA51 recA441 defective cells, the spontaneous yield of these mutations is elevated and UV irradiation produces a significant decrease in the numbers of this particular mutation. As explanation we suggest that the spontaneous appearance of these mutations reflects mutation at apurinic sites, the efficiency of which is elevated in lexA51 recA441 cells (with derepressed SOS functions and an activated form of RecA protein). The addition of UV damage in the DNA of these cells cannot further stimulate the positive functions that are required for the production of these mutations and are typically associated with UV mutagenesis (induction of SOS functions, activation of RecA protein and introduction of a targeting photoproduct) but apparently can have a negative effect on mutagenesis, hitherto not realized.     

Mutagenic DNA repair in Escherichia coli XIII Proofreading exonuclease of DNA polymerase III holoenzyme is not operational during UV mutagenesis

We have introduced a mutD5 mutation (which results in defective 3′–5′-exonuclease activity of the ϵ proofreading subunit of DNA polymerase III holoenzyme) into excision-defective Escherichia coli strains with varying SOS responses to UV light. MutD5 increased the spontaneous mutation frequency in all strains tested, including recA430, umuC122::Tn5, and umuC36 derivatives. It had no effect of UV mutability or immutability in any strain or on misincorporation revealed by delayed photoreversal in UV-irradiated umuC36, umuC122::Tn5, or recA430 bacteria. It is concluded that the ϵ proofreading subunit of DNA polymerase III holoenzyme is excluded, inhibited, or inoperative during misincorporation and mutagenesis after UV.     

Thermal resistance to photoreactivation of ultraviolet light induced mutations in the lacI gene of E. coli ung

Ultraviolet light (UV) induced mutations in the lacI gene of Escherichia coli are thought to be targeted by DNA photoproducts. A number of reports suggest that both cyclobutyl pyrimidine dimers and pyrimidine (6−4) pyrimidone photoproducts may be involved. To investigate the potential contribution of each of these DNA photoproducts to mutagenesis in the lacI gene, we held UV-irradiated cells at a temperature of 44°C for 75 min and then exposed them to photoreactivating light (PR). This protocol is expected to preferentially deaminate specifically those cytosines that are contained in cyclobutyl dimers and subsequently monomerize the dimers to yield uracils in the DNA. In a strain deficient for uracil-DNA glycosylase (Ung⁻), these uracils would not be removed and a G : C → A : T transition would result at the site of the dimer. This protocol resulted in the enhancement of amber nonsense mutations that result from transitions at potential cytosine-containing dimer sites. The enhanced mutation frequencies resulting from this procedure were used to estimate the probability of dimer formation at the individual sites. A comparison of the dimer distribution with the mutation frequencies following UV alone suggests that both cyclobutyl dimers and (6−4) photoproducts contribute to UV-mutagenesis in the lacI gene. In addition, we conclude that the frequency of mutation at any particular site not only reflects the occurrence of DNA damage, but also the action of metabolic processes that are responsible for DNA repair and mutagenesis.     

umuC-dependent and umuC-independent gamma- and UV-radiation mutagenesis in Escherichia coli

The effects of the umuC36 and umuC122::Tn 5 mutations on gamma- and UV radiation mutagenesis (nonsense, missense, and frameshift mutation assays) in Escherichia coli K12 were studied. Although both mutations reduced radiation mutagenesis, the umuC36 mutation appeared to be leaky since considerably more UV radiation mutagenesis could be detected in the umuC36 strain than in the umuC122::Tn 5 strain. In general, the umuC strain showed much larger deficiencies in UV radiation mutagenesis than they did for gamma-radiation mutagenesis. The mutability of the umuC122:: Tn 5 strain varied depending on the radiation dose, and the mutation assay used. For gamma-radiation mutagenesis, the deficiency varied from no deficiency to a 50-fold deficiency; for UV radiation mutagenesis, the deficiency varied from 100-fold to at least 5000-fold. We concluded that both umuC-dependent and umuC-independent modes function for gamma-radiation mutagenesis, while UV radiation mutagenesis seems to depend almost exclusively on the umuC-dependent mode.     

Differential repair of premutational UV-lesions at tRNA genes in E. coli.

Summary Ultraviolet radiation produces bacterial revertants that frequently are the result of suppressor mutation. When irradiated cells are incubated under conditions unfavorable for protein synthesis there may be a large decrease in the frequency of observed mutants (mutation frequency decline, or MFD). MFD occurs only in excision-proficient strains and is inhibited by inhibitors of pyrimidine dimer excision. It has therefore been interpreted as enhanced excision of some premutational lesions. Potential de novo UAG suppressor mutation is very susceptible to MFD. Potential conversion mutation, the conversion of a UAG to a UAA suppressor, is at least ten times less susceptible to MFD. A base pair transition at a GC target in a particular tRNA gene is suggested for both de novo suppressor mutation and for conversion mutation. We interpret these results as indicating differential repair of premutational UV photoproducts at two closely spaced sites in the same tRNA gene. The significant difference between these two types of mutation may be the orientation of this target base pair in double helical DNA. The C would be in the transcribed strand of DNA when a nucleic acid alteration produces de novo suppressor mutation. The C would be in the nontranscribed strand, two base pairs removed, when a mutagenic alteration produces suppressor conversion. A model involving facilitated incision by hybridization of the transcribed strand of DNA to its cognate tRNA, under conditions promoting MFD, is described to explain this differential repair.     

Escherichia coli umuDC mutants: DNA sequence alterations and UmuD cleavage

Summary The products of the chromosomally encoded umuDC genes are directly required for mutagenesis in Escherichia coli. Strains with either umuD or umuC mutations are rendered phenotypically non-mutable. To ascertain the molecular basis of this non-mutability, we determined the DNA sequence alterations of seven chromosomal umuDC mutants. Six mutants (umuD1, umuD44, umuD77, umuC36, umuC25, and umuC104) were found to be single base-pair substitutions that resulted in missense mutations. The Tn5 transposon insertion mutation (umuC122) resulted in a missense mutation followed immediately by a termination codon, producing a truncated UmuC protein lacking 102 carboxyl-terminal amino acids. All of the mutations were found to reside in regions of the UmuD and UmuC proteins that share high homology with analogous proteins. Chemiluminescent immunoassays revealed that the umuD1, umuD44, and umuD77 mutations all resulted in a non-cleavable UmuD protein. Because UmuD cleavage is a prerequisite for mutagenesis, the lack of UmuD processing appears to be the molecular basis for the non-mutable phenotype in these strains. These studies re-emphasize the critical nature of the RecA-mediated cleavage of UmuD for inducible mutagenesis and provide insights into the functional domains of the UmuC protein.