Reversion of argE3 to Arg + in Escherichia coli AB1157 — an

This review concerns reversion of the argE3 (ochre) nonsense mutation to prototrophy in E. coli AB1157 strain as an informative system for mutation detection. Strain AB1157 bears the argE3 (ochre), hisG4 (ochre) and thr-1 (amber) mutations, and the supE44 amber suppressor on its chromosome. The Arg(+) phenotype can be restored by (i) any base substitution at the argE3 site that changes the nonsense UAA codon to any sense nucleotide triplet or to UAG recognized by the supE44 amber suppressor, or (ii) suppressor mutations enabling the reading of the UAA nonsense codon. The argE3 → Arg(+) reversion-based system enables (i) determination of the spontaneous or induced mutation level; (ii) determination of base substitutions (suppressor analysis); (iii) examination of transcription-coupled repair (TCR) since targets for DNA damage are situated on the transcribed or coding strand of DNA; (iv) detection of mutations resulting from single stranded DNA damage. This review focuses on studies carried out since the early 1990s till now with the application of the AB1157-based mutation detection system. Recently, the system has been used to obtain new data on the processes of methyl methanesulfonate-induced mutagenesis and DNA repair in E. coli alkB⁻ mutants.


INTRoDUCTIoN
Bacteria are simple and widely used models for examination of mutagenesis and DNA repair processes.Two representatives of Enterobacteriaceae, Escherichia coli and Salmonella typhimurium, are commonly used in studies on spontaneous and induced mutagenesis.The advantages of bacterial systems are their availability, ease of cultivation, short time of cell division, and haploidy.Many DNA-damaging agents and/or mutator genes cause mutations that are readily and clearly observable as changes of phenotype.Additional observations like (i) analysis of bacterial survival after treatment with mutagenic agents; (ii) microscopic examination of bacterial cells; (iii) induction of the SOS system measured by induction of b-galactosidase from umuC::lacZ fusion; (iv) examination of plasmid DNA isolated from MMS-treated cells for their sensitivity to the specific endonucleases Fpg and Nth that recognize abasic sites, all provide a simple and rapid yet highly informative characterization of the examined processes.

EschErichia coli K12 AB1157 model
Escherichia coli K12 was isolated from the stool of a convalescent diphtheria patient in the US (Palo Alto, California) in 1922 and deposited in the strain collection of the Department of Bacteriology of Stanford University.Many mutant derivatives of strain K12 have been obtained in numerous laboratories around the world.One of them is strain AB1157 with the relevant genotype: thr-1 ara-14 leuB6 D(gpt-proA)62 lacY1 tsx-33 supE44 amber, galK2 hisG4 rfbD1 mgl-51 rpsL31 kdgK51 xyl-5 mtl-1 argE3 thi-1 and its derivatives (Bachman, 1987).The argE3 (ochre), hisG4 (ochre) and thr-1 (amber) are nonsense point mutations in genes encoding enzymes involved in arginine, histidine and threonine biosynthesis, respectively.supE44 encodes the supE amber suppressor reading UAG.However, it can only weakly suppress the thr-1 mutation.
A suppressor mutation is a mutation that counteracts the effects of another mutation.One type of suppressor mutations are mutations that appear in the tRNA-encoding genes at the anticodon site.The changed tRNAs are able to recognize a nonsense codon that occurs elsewhere in protein-coding genes and incorporate the amino acids specific for them into the polypeptide chain during protein synthesis.
The bacterial test system of mutation detection described here is based on reversion of the auxotrophic argE3 mutation to prototrophy.The marker is situated in the chromosome.The Arg + phenotype can be restored by (i) any point mutation at argE3 that changes the nonsense UAA codon to any sense nucleotide triplet coding for any amino acid; (ii) an AT→GC transition at argE3 that changes the UAA nonsense codon to the UAG nonsense codon recognized by the supE44 amber suppressor; and (iii) suppressor mutations enabling reading of the UAA nonsense codon.The suppressors can be created de novo or as a result of a GC→AT transition at supE44 (formation of supE ochre suppressor) (Sargentini & Smith, 1989;Śledziewska-Gójska et al., 1992).
Considering all the theoretical possibilities of ochre suppressor formation in E. coli resulting from a single base substitution in tRNAs genes, it can be seen that such suppressors may arise from tRNA for tyrosine, lysine, glutamine, glutamate, leucine and serine.The fol-lowing tRNA species that may produce de novo an ochre suppressor by a single base substitution in the anticodon site are tRNA Gln UUG , tRNA Lys UUU , and tRNA Tyr GUA .The formed suppressors are, respectively, supB, supL (supG, supN) and supC (supO, supM), created as a result of GC→AT (supB), AT→TA (supL, supG, supN) or GC→TA (supC, supO, supM) base substitutions in gln-tRNA, lys-tRNA and tyr-tRNA genes, respectively (Table 1).The supX suppressor is also found in the Arg + revertants, but it has not yet been identified.This suppressor can be formed as a result of either GC→TA or AT→TA transversions (Sargentini & Smith, 1989;Śledziewska-Gójska et al., 1992, and cited therein).Raftery and Yarus (1987) constructed the gltT(SuUUA/G) gene encoding tRNA Glu UUA as a result of a GC→TA transversion in the gltT gene encoding tRNA Glu UUC .This construct was expected to explain the mystery of the supX suppressor.However, it failed to suppress the argE3 ochre mutation in E. coli AB1157 strain (Płachta & Janion, 1992).Moreover, Prival (1996) identified three tRNA Glu  UUA suppressors: supY, supW and supZ that arose from the gltW, gltU and gltT genes, respectively.These suppressors were found in late-arising spontaneous Arg + revertants.
There are also theoretical possibilities of creating ochre suppressors from tRNA Tyr AUA , tRNA Ser UGA and tR-NA Leu  UAA , but these suppressors have not been identified yet (Śledziewska-Gójska et al., 1992).Figure 1 shows two schematic pictures of tRNA suppressors.
Arg + revertants can arise spontaneously or as a result of induced mutagenesis.The first step in the analysis of the Arg + revertants is the examination of their requirement for histidine and threonine for growth.Arg + revertants have been divided into four phenotypic classes (class I: Arg + His -Thr -, class II: Arg + His + Thr -, class III: Arg + His -Thr + and class IV: Arg + His + Thr + ).Because, as mentioned above, supE44 suppresses to some extent the thr-1 mutation, the Thr + phenotype may be wrongly read and thus the revertants of class I and class II may be incorrectly classified as class III and class IV, respectively.For this reason in practice only two groups of Arg + revertants have usually been considered: a sum of classes I and III, and a sum of classes II and IV (Todd et al., 1979;Śledziewska-Gójska et al., 1992).
The sensitivity of the Arg + revertants to tester T4 phages is the second step in mutational analysis.A set of five T4 phages carrying a defined nonsense mutation includes the following phage mutants: amber B17 and NG19, and ochre oc427, ps292 and ps205.Phage multiplication, observed as plaque formation on a lawn of tested bacteria, indicates that the host bacterium bears a specific suppressor mutation (Kato et al., 1980;Shinoura et al., 1983;Sargentini & Smith, 1989;Śledziewska-Gójska et al., 1992).
Arg + revertants of class I are the result of back mutations at the argE3 site, or supB or supE ochre suppressor formation.Arg + revertants of class II, III and IV occur as a result of supL, supX and supC suppressor formation, respectively.The details of the above analysis are contained in Table 1, in which species of tRNA producing the indicated suppressor by a single base substitution in the anticodon sites are also included.Sargentini and Smith (1989) constructed a set of AB1157 derivatives bearing all the mentioned suppressors.
Identification of created suppressors allows deducing the specificity of mutation without DNA sequencing.However, such analysis does not say anything about the type of mutations in the argE gene creating a sense codon from the UAA stop codon.In this case DNA sequencing is required.The proportion of suppressor and It is also possible to study the level of hisG4→His + revertants; however, only some suppressors may counteract the effect of the hisG4 mutation so that many of the arising mutations are lost (Śledziewska-Gójska et al., 1992).

StudieS with the uSe oF the arge3→Arg + ReveRSion-BASed SyStem
In the era of intensive development of techniques of molecular biology and genetics studies based on reversion to prototrophy of the argE3 mutation still provide new, interesting and valuable information: not only have they confirmed current knowledge but also supplied new data on the mutagenic specificity of different mutagens and mutator genes as well as on the mechanisms of mutagenesis and DNA repair.The applications of the described genetic system are presented below.This system is particularly useful for detection of GC→TA, GC→AT and AT→TA base substitutions and examination of transcription-coupled DNA repair.
DNA polymerase III, the main replicative polymerase in E. coli, comprises a dnaQ-encoded epsilon subunit responsible for proofreading activity.Mutants defective in this subunit chronically express the SOS response and exhibit a mutator phenotype (Echols et al., 1983).Using the argE3→Arg + reversion, the effects of deletions in genes polB and umuDC, encoding, respectively, the SOSinduced DNA polymerases Pol II and Pol V, on the frequency and specificity of spontaneous mutations in the dnaQ background were studied.It was clearly shown that deletion of umuDC genes significantly decreased the level of spontaneous mutations in dnaQ strains (Nowosielska et al., 2004a).The Arg + revertants in mutD5 (allele of dnaQ) mutant occurred only as a result of tRNA suppressor formation, whereas those in mutD5 polB (Pol II deficient) strains arose at 81 % by back mutation at the argE3 ochre site (Nowosielska et al., 2004b).
A mutator phenotype measured by an increased level of spontaneous umuDC-dependent argE3→Arg + reversions was one of the symptoms of the chronic induction of the SOS system in a nth xth nfo mutant deficient in base excision DNA repair system (BER).This mutant can not repair abasic sites.The chronic induction of the SOS system is due to accumulation of abasic sites left unrepaired in DNA (Janion et al., 2003).
Studies on E. coli AB1157 strain and its derivatives revealed that biological effects (survival, mutation induction and mutation specificity) of halogen light irradiation were very similar to those observed after UVC irradiation.The halogen light-induced mutations were GC→AT transitions (supB or supE ochre suppressor formation) and back mutations at argE3 sites resulting from T-C 6-4 photoproducts or T<>T thymine dimers, respectively.The latter damage was observed only in uvrA mutants defective in nucleotide excision repair (NER), constituting less than 5 % of the total number of Arg + revertants (Wójcik & Janion, 1997;1999).Those results confirmed previous data showing that halogen light causes harmful effects such as DNA damage, mutations, genotoxicity and skin cancers in mice due to emission of a broad spectrum of UV light, particularly UVC (De Flora et al., 1990;D'Agostini et al., 1993;D'Agostini & De Flora, 1994).
Analysis of Arg + revertants supplied new data on the mechanisms of mutagenesis and processes of DNA repair.The mutagenic properties of DNA damaging agents and the spectra of the induced mutations depend on the bacterial background, i.e., the presence of mutations in genes encoding proteins involved in DNA repair systems.
It was known that EMS was an umuDC-independent mutagen and induced GC→AT transitions due to formation of O 6 -ethylguanine in DNA.It has been shown that in the AB1157 strain, EMS-induced Arg + revertants arise by supB and supE ochre suppressor formation.However, in mutS -, a mismatch repair-deficient strain, the specificity of the EMS-induced argE3→Arg + reversions was changed and formation of supL suppressor by AT→TA transversions was mainly observed.Moreover, these mutations were umuDC-dependent.It was suggested that the change in mutation specificity was due to 3meA lesions or creation of apurinic sites.These results also point to different processes of DNA repair in mutS + and mutS -strains (Grzesiuk & Janion, 1993).
The spectrum of the MMS-induced argE3→Arg + reversions changes in various strains deficient in DNA repair systems.In the mutS -mutant Arg + revertants arose mainly by GC→AT transitions (supB and supE ochre suppressor formation) or back mutations at argE3 site.The latter group constituted a few percent of the total number of the Arg + revertants (Grzesiuk & Janion, 1998).In the dnaQ49 derivative of the AB1157 strain about half of the MMS-induced Arg + revertants occurred by AT→TA transversions (supL suppressor formation).In a double dnaQ -umuDC -mutant about 90 % of the revertants possessed supB or supE ochre suppressors due to GC→AT transitions (Grzesiuk & Janion, 1996)

Detection of mutations resulting from lesions in ssDNA
Examination of MMS-induced mutagenesis in AB1157alkB -derivatives indicates that the argE3→Arg + reversion system also enables detection of mutations arising from lesions in ssDNA (Nieminuszczy et al., 2006a;2009;Sikora et al., 2010;Wrzesiński et al., 2010).AlkB is an α-ketoglutarate-, O 2 -and Fe(II)-dependent dioxygenase that oxidatively demethylates 1meA and 3meC in dsand ssDNA and in RNA.However, ssDNA is repaired much more effectively than dsDNA (Trewick et al., 2002;Falnes et al., 2002).It has been shown that in alkB -mutants the level of MMS-induced mutagenesis depends on the test system used, and is several orders of magnitude higher when measured in the argE3→Arg + reversion test system in E. coli AB1157 in comparison to lacZ→Lac + reversion studied in CC101-CC106 strains (Nieminuszczy et al., 2006a;2006b;2009;Kataoka et al., 1983;Dinglay et al., 2000).The CC101-CC106 tester strains are described in more detail in Chapter: Other enterobacterial systems for mutation detection.Briefly, the lacZ→Lac + reversion occurs only by a back mutation at one point in the structural gene encoding the β-galactosidase that if not expressed would be primarily in dsDNA form.
The argE3→Arg + reversion-based system showed that in AB1157 alkB -strain 95-98 % of the induced mutations were umuDC (Pol V)-dependent AT→TA transversions (supL suppressor formation) and GC→AT transitions (supB or supE ochre suppressor formation).Back mutations in the argE3 site constitute only about 2-5 % of all types of Arg + revertants (Nieminuszczy et al., 2006a).Genes encoding tRNA are heavily transcribed and exist mostly as ssDNA in cells.It facilitates methylation of A/C to 1meA/3meC.That is why we assume that in AB1157 alkB -strain the targets undergoing mutations leading do Arg + revertants are predominantly located in ssDNA.Reversion to Arg + occurs mostly by formation of a variety of suptRNA ochre suppressors.The number of targets undergoing mutations and differences in the reactivity of MMS to form 1meA/3meC lesions in ssDNA vs. dsDNA are the main reasons of the great discrepancy in the frequencies of MMS-induced argE3→Arg + and lacZ→Lac + revertants observed (Nieminuszczy et al., 2009).
In strains deficient in the BER system (abasic site repair), mutants in nfo (encoding endonuclease IV) and xth (encoding exonuclease III) genes with non-functional AlkB protein, an extremely high level of the MMS-induced argE3→Arg + reversions has been observed.This phenomenon can be explained by the involvement of abasic sites formed in DNA of the AB1157 nfo -xth - strain, causing a local relaxation of dsDNA structure.We argE3Arg + reversion in E. coli AB1157 as a system for mutation detection assume that under these conditions more single-stranded DNA appears in the bacterial chromosome that faciliates methylation by MMS and results in "error catastrophy" in the triple AB1157 nfo xth alkB mutant (Sikora et al., 2010).Analysis of MMS-induced Arg + revertants in alkB -and alkB -BER -strains clearly points to a mutagenic activity of 1meA and 3meC (Nieminuszczy et al., 2006a;Sikora et al., 2010).

Determination of transcription-coupled DNA repair
The argE3→Arg + reversion system of mutation detection in E. coli AB1157 also enables studies on preferential removal of lesions from the transcribed strand of DNA.This type of DNA repair, called transcription-coupled DNA repair (TCR), requires Mfd protein that removes transcription elongation complexes stalled at non-coding lesions in DNA and recruits to these sites proteins involved in nucleotide excision repair (NER).TCR occurs under conditions of temporary inhibition of protein synthesis and results in a decrease in the frequency of induced mutations (Selby & Sancar, 1993;Savery, 2007).This phenomenon is called mutation frequency decline (MFD) and was discovered for UV-irradiated bacteria by Evelin Witkin (for review see Witkin, 1994).The MFD phenomenon has been studied by the Janion and Grzesiuk's group on UV (or halogen light)-and MMS-induced Arg + revertants in the AB1157 strain transiently incubated under non-growth conditions (amino acid starvation) after treatment with a mutagen (Grzesiuk & Janion, 1994;1996;1998;Wójcik & Janion, 1997;Fabisiewicz & Janion, 1998;Wrzesiński et al., 2010).Table 2 shows all the mutagenic targets for UV-and MMS-induced DNA damage.Potential targets for UV-modifications (T-C and T-T sequences for creation of 6-4 photoproducts and pyrimidine dimers, respectively) are underlined.Potential targets (single bases) for MMS-modifications are shadowed.UV-or halogen light-induced Arg + revertants occur mainly as a result of a GC→AT transition forming the supB and supE ochre suppressors, respectively, at the transcribed DNA strand of the glnU and the coding DNA strand of the glnV amber (supE44 amber) gene.In mfd -strains the formation of supB predominated over supE ochre suppressors and their number, in contrast to the mfd + strain, did not decrease during amino acid starvation.The MFD effect observed in mfd + strains is a reflection of repair of premutagenic lesions in the transcribed strand of the glnU gene leading to supB suppressor formation (Wójcik & Janion, 1997;Fabisiewicz & Janion, 1998).
Studies on the MFD effect in the AB1157 strain and its derivatives after MMS treatment involved both an analysis of Arg + revertants and examination of plasmid DNA isolated from cells treated with MMS for their sensitivity to the Fpg and Nth endonucleases.The decrease in the level of MMS-induced mutations during transient starvation was accompanied by repair of abasic sites in plasmid DNA.As it is shown in Table 2, potential targets for MMS damage are located on both the transcribed and coding DNA strands of glnU, glnV amber and argE genes and only on the transcribed strand of lys-tRNA genes.Lesions resulting from methylation of the transcribed DNA strand are subject to MFD repair.Previous studies on the MFD phenomenon after MMS treatment of the AB1157 strain and its derivatives focused on the preferential repair of transcribed-strand lesions of genes coding for lys-tRNA; this repair was manifested by a decrease in the number of supL suppressors (Grzesiuk & Janion, 1994;1998).A recent study revealed a significantly slower and completely absent MFD effect in, respectively, AB1157mfd and double alkB mfd mutants.It was assumed that the former effect is the result of action of other DNA repair systems and the latter is a reflection of an accumulation of DNA damage and strong SOS induction.These results again have confirmed the strong mutagenic effects of 1meA/3meC lesions (Wrzesiński et al., 2010).
Interestingly, in a dnaQ mutant no MFD repair was observed indicating that in this mutant the processes of DNA repair are different, probably due to chronic induction of SOS response and the presence of Pol V and Pol IV DNA repair polymerases induced within this response (Grzesiuk & Janion, 1996).

Adaptive mutation
Reversion to prototrophy of the argE3 mutation was also used as a method for determination of adaptive mutations.These mutations (also called "stationary phase" or "starvation associated") are a special kind of spontaneous mutations that occur in non-dividing cells and allow growth under selected conditions.Using the argE3→Arg + reversion system, AB1157 bacteria that mutated adaptively were selected on minimal plates devoid of arginine after 4 to 10 days of growth.Further phenotypic analysis and susceptibility to a set of amber and ochre T4 phages allowed the identification of stationary phase mutations in mutY - (Nowosielska & Grzesiuk, 2000) and dnaQ - (Nowosielska et al., 2004a;2004b) strains.It has been shown that in the dnaQ -strain Pol IV and Pol V polymerases, in contrast to Pol II, influence the frequency and specificity of starvation-associated mutations.

oTHeR eNTeRoBACTeRIAl SySTeMS foR MUTATIoN DeTeCTIoN
Reversions of trpE65 to Trp + phenotype and tyrA14 to Tyr + in E. coli B/r WP2 (Ohta et al., 2002) and WU3610 derivatives (Bockrath et al., 1987), respectively, are analogous mutation detection systems to that of E. coli K12 AB1157.Both trpE65 and tyrA14 are ochre mutations in genes coding for enzymes involved in tryptophane and tyrosine biosynthesis, respectively.The Trp + or Tyr + phenotype may be recovered by (i) any point mutation at trpE65 or tyrA14 leading to the formation of a sense nucleotide triplet, and (ii) ochre suppressor mutations.In the WP2 (trpE65) system the examined suppressors are supB, supC, supG and supM formed in the genes coding for tRNA: glnU, tyrT, lysT and tyrU, respectively (Ohta et al., 2002).In the WU3610 (tyrA14) strain the de novo ochre suppressor mutations in glutamine tRNA are studied.The WU3610-11 derivative bears an amber suppressor created from another glutamine tRNA gene that can be converted to an ochre suppressor (Bockrath & Palmer, 1977;Bockrath et al., 1987).Both systems have been used in MFD studies (Bridges et al., 1967;George & Witkin, 1974;Bockrath & Palmer, 1977;Bockrath et al., 1987).
A commonly used bacterial system allowing rapid detection of specificity of mutation is the lacZ→Lac + reversion in E. coli K12 CC101-111 strains.The lacZ gene encodes b-galactosidase and is part of the lactose operon.Mutants in lacZ gene are unable to grow on a medium containing lactose as the sole carbon source.A set of eleven mutants with a lacZ deletion in the chromosome and F' with cloned lacZ gene bearing defined mutations (six base substitutions and five frame shifts at the same coding position) have been constructed.Reversion to the Lac + phenotype is due to a specific mutation in the lacZ gene on F' (Coupples & Miller, 1989;Coupples et al., 1990).In this case the marker is episomal, in contrast to the AB1157 strain where the marker is situated on the chromosome.
E. coli K12 FC40 is another mutation detection system based on lacZ→Lac + reversion.This strain contains an F' episome carrying a lacI-lacZ fusion with a +1 base pair frameshift mutation, lacI33, in the lacI coding sequence.The Lac -→Lac + reversion is due to a -1 frameshift mutation.FC40 was often used in studies on stationaryphase mutations (Foster, 1994).
The Salmonella mutagenicity assay was introduced by Bruce Ames and co-workers in the early 1970s and later modified (Ames et al., 1973).Briefly, the test involves reversion of a his -auxotrophic mutation to prototrophy by base substitutions in the hisG46 allele or by frameshift in the hisD3052 allele.In addition, the tester strains carry: (i) additional mutations, such as rfa, increasing the permeability of the bacterial cell wall and enabling better penetration of mutagenic agents to the cell, or uvrB, disturbing DNA repair; (ii) plasmid pKM101 -a mutagenesis-enhancing plasmid bearing mucA and mucB genes that code for proteins corresponding to E.coli UmuC and UmuD, responsible for translesion synthesis (TLS) (Mortelmans, 2006).
Antibiotic resistance to streptomycin, rifampicin or nalidixic acid are also often used markers for determination of the level of spontaneous and induced mutations in bacteria.Rifampicin-, streptomycin-and nalidixic acidresistant mutants arise following mutations in the rpoB gene encoding β subunit of RNA polymerase, mutations leading to changes in the 30S subunit of the ribosome or mutations in genes encoding gyrase subunits, respectively.

Figure 1 .
Figure 1.How mutations in the anticodon create suppressor tRNAs (A) Tyrosine-specific tRNA recognizing the UAC codon in mRNA changes into supC suppressor reading UAA as a result of GC→TA transversion in the tyrT gene.Anticodon triplets are shown in blue and red for tyrT tRNA and supC suppressor, respectively.(B) Amber suppressor tRNA, supE amber (glnV amber), arising from the glnV gene (encoding tRNA for glutamine) recognizing the UAG codon in mRNA changes into supE ochre suppressor (reading UAA) as a result of GC→AT transition in the supE amber (glnV amber) gene.Anticodon triplets are shown in blue and red for supE amber (glnV amber) and supE ochre, respectively.