Mutation frequency decline in Escherichia coli B/r after mutagenesis with ethyl methanesulfonate

Nonsense-defective auxotrophic strains of Escherichia coli B/r were used to study mutation frequency decline (MFD) after mutagenesis with ethyl methanesulfonate (EMS). The mutation frequencies for prototrophic revertants that were either converted or de novo glutamine tRNA suppressor mutations declined as treated auxotrophic parental cells were incubated with glucose but without required amino acids (a condition typically producing MFD). The decline for converted suppressor mutations was more rapid than the decline for de novo suppressor mutations after low or moderate EMS treatment, but both suppressor mutation types showed the same slow decline after extensive treatment. The declines for both types of suppressor mutation were eliminated in uvrA-defective cells, and the rapid decline seen for converted suppressor mutations appeared as a slow decline in mfd-defective cells. The results are interpreted that true MFD (the rapid process) affects only the EMS-induced converted glutamine tRNA suppressor mutations. This would account for the rapid decline that is blocked in cells with an mfd defect and in cells with deficient excision repair activity (uvrA or excessive DNA damage). In addition, a second non-specific antimutation mechanism is proposed that is dependent on excision repair only and accounts for the slow decline seen with converted suppressor mutations in some instances and with de novo suppressor mutations at all times. The true MFD mechanism may consist of a physiologically dependent facilitated excision repair specifically for premutational residues located in the transcribed strand of the target DNA sequence (for O6-ethylguanine in cells treated with ethyl methanesulfonate or pyrimidine-pyrimidine photoproducts after UV irradiation).     

UV-mutagenesis at a cloned target sequence: converted suppressor mutation is insensitive to mutation frequency decline regardless of the gene orientation

Premutational lesions produced by ultraviolet radiation in the Gln2 tRNA genes of E. coli B/r show differing sensitivities to a mutation avoidance phenomenon known as mutation frequency decline (MFD). A mutation event that changes the wild-type gene to an amber (UAG) suppressor is normally sensitive to MFD. Mutation of this amber suppressor to an ochre (UAA) suppressor is not sensitive to MFD. These two mutation events occur in the same anticodon region of the DNA. The dissimilarity of MFD sensitivity between these two mutations may result because the respective premutational photoproducts for the two are located in opposite strands of duplex DNA. To examine the effect of strand position of the premutational lesions on MFD, recombinant lambda phage were constructed that contained the amber suppressor as a mutation target in the two possible orientations. Comparison of MFD in bacterial lysogens containing either of the two types of recombinant prophage indicated that reversing the orientation of the target sequence relative to adjacent bacterial DNA had no effect on MFD. Since rotational inversion of the target sequence did not alter the sensitivity to MFD of mutation occurring at the cloned target gene, the antimutation process inherent to MFD can not be attributed to an asymmetrical interaction between the template strands and the DNA-replication complex.     

Ultraviolet photoproducts at the ochre suppressor mutation site in the glnU gene of Escherichia coli: relevance to "mutation frequency decline"

Summary Ochre suppressor mutations induced by UV in the Escherichia coli glnU tRNA gene are CG to TA transitions at the first letter of the anticodon-encoding triplet, CAA. Premutational UV photoproducts at this site have long been known to exhibit an excision repair anomaly (mutation frequency decline or MFD), whereby post-irradiation inhibition of protein synthesis enhances their excision and reduces suppressor mutation yields ten-fold. We sought to clarify the basis of this unique repair response by determining the spectrum of UV photoproducts on both strands of a 36 by region of glnU which includes the anticodon-encoding triplet. We found that four different photolesions are produced within the 3 by sequence corresponding to the tRNA anticodon: (i) on the transcribed strand, TC (6–4) photoproducts and TC cyclobutane dimers are formed in equal numbers at the site of the C to T transition, indicating that this site is a hotspot for the usually less frequent (6–4) photoproduct; (ii) on the nontranscribed strand, TT dimers are found opposite the second and third letters of the anticodon-encoding triplet, adjacent to the mutation site; and (iii) on the nontranscribed strand, an alkali-sensitive lesion other than a (6–4) photoproduct is formed, apparently at the G in the mutation site. We suggest that mutation frequency decline may reflect excision repair activity at closely spaced UV lesions on opposite strands, resulting in double-strand breaks and the death of potential mutants.     

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.     

Suppression of Glutamic Acid Codons by Mutant Glycine Transfer Ribonucleic Acid

In previous mutational studies with mutant trpA46 (Gly [GGA] --> Glu [GAA] at position 211 of the tryptophan synthetase alpha chain) of Escherichia coli, no missense suppressors were detected. Such suppressors have now been obtained by single mutations in gly Vins, the structural gene for a GGA/G-reading, mutationally altered form of gly V transfer ribonucleic acid (tRNA) (tRNA(Gly) which reads GGU/C). A trpA46 strain containing the gly Vins alteration was mutagenized with hydroxylamine, and suppressor mutations were detected in the prototrophs obtained. Eighteen independent suppressors were examined and shown to have alterations which map in the gly V region. Chromatography of the glycyl-tRNAs of one suppressed mutant on a benzoylated diethylaminoethyl-cellulose column revealed an alteration in the tRNA(ins) (Gly) peak. The trpA46 suppressor mutation thus appears to involve a change of tRNA(ins) (Gly) from a GGA/G (Gly) reader to a GAA (Glu) reader. Since this suppressor presumably retains the "wobble" pairing of gly Vins tRNA, it was used to select the conversion of GAU (Asp211) to GAG (Glu211) in the alpha chain. supD (serine-inserting amber suppressor) was then used to obtain the conversion of GAG (Glu211) to UAG211. Missense revertants of trpA (UAG211) are being isolated as a means of introducing new codons which can be used in the selection of additional missense suppressors.     

Mutagenic DNA repair in escherichia coli. V. Mutation frequency decline and error-free post-replication repair in an excision-proficient strain.

Mutation frequency decline (MFD) is an irreversible loss of newly-induced suppressor mutations occurring in excision-proficient Escherichia coli during a short period of incubation in minimal medium before plating on broth- or Casamino acids-enriched selective agar. It is known that MFD of UV-induced mutations may occur before DNA containing pre-mutagenic lesions is replicated, but we conclude that MFD can also occur after the damaged DNA has been replicated on the basis of the following evidence. (1) Mutation fixation in rich medium (i.e., loss of susceptibility to mutation frequency decline) with ethyl methanesulphonate mutagenesis begins immediately, whereas with UV it is delayed for 20--30 min. (2) The delay in mutation fixation after UV can be explained neither by inhibition of DNA replication nor by a delay in the appearance of error-prone repair activity in the irradiated population. (3) MFD at later times after UV irradiation is more rapid and is less strongly inhibited by caffeine than is MFD immediately after irradiation. (4) Excision is virtually complete 20 min after 3 J m-2 UV but at that time virtually all mutations are still susceptible to MFD. We have presented evidence elsewhere that in bacteria there is an alternative error-free excision-dependent type of post-replication repair of potentially mutagenic daughter strand gaps. We suggest that this process is inhibited at tRNA loci in the presence of nutrient broth or Casamino acids, possibly because of a broth-dependent change in the structure of the single-stranded region including the tRNA locus.     

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.     

Thermal resistance to photoreactivation of specific mutations potentiated in E. coli B/r ung by ultraviolet light

Summary Mutagenesis by ultraviolet light was studied in a strain of E. coli ung, which lacks uracil-DNA glycosylase activity. Mutation potentiated by UV in cells already induced by nalidixic acid treatment was still photoreversible suggesting that pyrimidine dimers act directly as premutational photoproducts. Secondly, irradiated cells were held in buffer at 48°C for 0 to 135 min to allow for deamination of cytosines in pyrimidine dimers. The mutation frequencies for class 2 de novo suppressor mutation, for class 2 converted suppressor mutation and for backmutation were individually determined, before and after photoreactivation, as a function of this thermal treatment. Backmutation remained sensitive to photoreactivation throughout the treatment but de novo and converted suppressor mutations rapidly developed resistance to photoreactivation. This resistance was not seen in an ung ⁺ control. A model is proposed to account for the selective resistance based on the hypothesis that class 2 de novo and converted suppressor mutations normally result from UV by GC to AT transitions at T=C dimers. The model describes deamination of the cytosine residues in these dimers to become uracil residues. In consequence, monomerization by photoreactivation in cells that can not repair uracils in DNA no longer reverses mutation and GC to AT transitions are established at the sites of uracils.     

Mutation frequency decline following chemical mutagenesis of Salmonella typhimurium

The occurrence is reported of a mutation frequency decline process (MFD) following treatment of Salmonella typhimurium strain trpC₃ with two chemical mutagens which give rise predominantly to suppressor revertants. With the carcinogen 4-nitroquinoline-N-oxide (4NQO) the results are analogous to those obtained for UV-mutagenesis. In the case of methoxynamine, the process is due to specific excision of premutational lesions, since lethality is low and lethal lesions are non-excisable. Mutants are described which cannot perform MFD of lesions induced by one or both of the chemical mutagens, indicating that the loss of revertants is in each case due to a bacteial repair system rather than to spontaneous degradation of the induced lesion. The mutants, however, were isolated because of an altered response to UV mutagenesis, viz., their ability to express UV-induced mutants in the absence of amino acids to stimulate active post-irradiation protein synthesis. In all other respects tested, their response to UV is identical with that of the parent strain. The hypothesis is discussed that the total absence of UV-induced revertants of the strain S. typhimurium trpC₃ when active protein synthesis is inhibited is due to two processes, first, rapid MFD due to the specific excision of pyrimidine dimers (the predominant UV-lesion) and secondly, the slow excision of other premutational damage which may be other photoproducts or secondary distortions caused by close juxtaposition of several pyrimidine dimers.     

De novo missense mutation Y174S in exon 1 of the adrenoleukodystrophy (ALD) gene

Adrenoleukodystrophy (ALD) is an X-linked disease, characterised by an alteration of the peroxisomal -oxidation of the very long chain fatty acids. The ALD gene has been identified and mutations have been detected in ALD patients. We report here a new missense mutation in the ALD gene of a male patient, predicting a tyrosine to serine substitution at codon 174 (mutation Y174S). The mother of the ALD patient does not have the Y174S mutation in her leukocyte DNA, indicating that Y174S arose de novo in the patient. Y174S is the first reported de novo mutation in the ALD gene.     

Fixation and loss of hydrazine-induced premutational damage in Haemophilus influenzae.

Premutational damage induced in Haemophilus influenzae by hydrazine appears to be fixed as final mutation only at replication as judged by the transformation assay. Fixation at replication is independent of the rec1 gene, unlike the case with nitrosocarbaryl. Prior to replication premutational damage induced by hydrazine disappears by an unknown process that is not dependent on the presence of a pyrimidine dimer excision system nor on the rec1 gene. Hydrazine does not produce detectable single-strand breaks or alkali-labile sites in the treated DNA nor gaps in DNA newly synthesized after treatment. In these respects it also differs from nitroso compounds. It is concluded that hydrazine acts to produce and altered base, possibly N(4)-aminocytosine, that produces mutations by mispairing at replication rather than by error-prone repair.