tRNA suppressors counteracting effects of the
Changes in genetic material result from introduction of mutations into DNA. Spontaneous mutations can occur because of replication errors or as a consequence of lesions introduced into DNA during normal cell growth. Induced mutations arise after treatment of the organism with an exogenous mutagen being physical or chemical agent increasing the frequency of mutations.
Bacteria are simple and widely used models for examination of mutagenesis and DNA repair processes. The advantages of bacterial systems are their availability, easy cultivation, short time of cell division, and haploidity. Many DNA damaging agents and/or mutator genes cause mutations that are readily and clearly observed in changes of phenotype. Additional observations like (i) analysis of bacterial survival after treatment with mutagenic agents; (ii) microscopic examination of bacterial cells; (iii) examination of plasmid DNAs isolated from mutagen-treated cells for their sensitivity to the specific enzymes that recognize DNA lesions; (iv) induction of the SOS system measured by induction of -galactosidase in
Two representatives of
2. Mutation detection systems in
argE3→ Arg+ reversion system in Escherichia coliK12
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 occur 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
Considering all the theoretical possibilities of the
There are also theoretical possibilities of creating ochre suppressors from tRNATyrAUA, tRNASerUGA and tRNALeuUAA, 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,
The sensitivity of 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, 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 and Smith, 1989; Śledziewska-Gójska et al., 1992). A schematic procedure for determination of MMS-induced mutagenesis using
Arg+ revertants of class I are the result of back mutations at the
|+||+||+||+||-||+||+ tS||+ tS||UAC||tyr||GC TA|
|+||-||+||-||-||+||-||+||?||?||GC TA or|
Sargentini and Smith (1989) constructed a set of AB1157 derivatives bearing all the mentioned suppressors: SR2151, SR2155, SR2162, SR2161, SR2154, SR2153 carrying, respectively,
Identification of created suppressors allows deducing the specificity of mutation without DNA sequencing. However, such analysis does not indicate the type of mutations in the
There is also a possibility to study the level of
2.2. 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, information on reversion to prototrophy of the
2.2.1. Specificity of mutator genes
The system confirms the mutagenic effects of mutator genes such as
MutT, MutY and Fpg (MutM), proteins belonging to the GO system, defend bacteria against the mutagenic action of 8-oxoG in DNA. MutT is a pyrophosphatase that hydrolyses 8-oxo-dGTP and prevents its incorporation into DNA. MutY is a DNA glycosylase excising from DNA adenine mispaired with A, 8-oxoG or G. Among others, Fpg excises from DNA 8-oxoG when it pairs with C (or T). The level of spontaneous transversions: AT→CG in
DNA polymerase III, the main replicative polymerase in
The SOS response is a bacterial defence system enabling the survival of cells whose DNA has been damaged and replication arrested. The SOS system increases expression of over 40 genes involved in DNA repair, replication, and mutagenesis. The expression of genes of the SOS regulon is tightly regulated. The
It has been shown that BW535 (
2.2.2. Specificity of mutagens
The mutagenic specificity of
Using this system it has been established that HA, a cytosine modifying agen, apart from well known GC→AT transitions may also cause a significant number of GC (or AT)→TA transversions. As much as 30% of the HA-induced Arg+ revertants were formed by GC (or AT)→TA transversions (Śledziewska-Gójska et al., 1992).
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). These results confirmed previous data showing harmful effects caused by halogen light, 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 is known that EMS, a SN2-type alkylating agent, is an umuDC-independent mutagen and induces GC→AT transitions due to formation of O6-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
MMS, another SN2-type alkylating agent, predominantly methylates nitrogen atoms in purines. This methylating agent creates the following adducts in double stranded DNA: 7-methylguanine (7meG), 3-methyladenine (3meA), 1-methyladenine (1meA), 7-methyladenine (7meA), 3-methylguanine (3meG), O6-methylguanine (O6meG), 3-methylcytosine (3meC), and methylphosphotriesters. In ssDNA, MMS induces the same lesions but in different proportions. In ssDNA, the participation of 1meA and 3meC increases significantly since the ring nitrogens at these positions are not protected by the complementary DNA strand (Wyatt & Pittman, 2006; Sedgwick et al., 2007). Analysis of Arg+ revertants in E. coli AB1157 strain without any additional mutations revealed that 70-80% of those revertants arose by AT→TA transversions in a umuDC-dependent process, whereas the rest occurred in a umuDC-independent manner either by GC→AT transitions (formation of supB or supE ochre suppressors) or by back mutations at argE3 site. The latter ones were detected in less than 5% of the Arg+ revertants. AT→TA transversions are thought to be the result of 3meA, abasic sites and 1meA, whereas GC→AT transitions come from O6-meG and 3meC residues in DNA and from depurination of 7meG (Grzesiuk & Janion, 1994; Nieminuszczy et al., 2006a; 2009; Wrzesinski et al., 2010).
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
2.2.3. 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-, O2- and Fe(II)-dependent dioxygenase that oxidatively demethylates 1meA and 3meC in ds- and 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
The argE3→Arg+ reversion-based system has showed that in AB1157 alkB– strain 95-98% of the induced mutations are 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
An extremely high level of the MMS-induced argE3→Arg+ reversions has been observed in E. coli AB1157 nfo xth alkB strain defected in the repair of AP sites caused by invalid base excision repair system (BER) and deficiency in AlkB dioxygenase. This phenomenon can be explained by local relaxation of dsDNA structure due to the presence of AP sites in AB1157 nfo
2.2.4. Determination of transcription-coupled DNA repair
The argE3→Arg+ reversion system in E. coli AB1157 also enables studies on preferential removal of lesions from the transcribed DNA strand. This type of DNA repair, called transcription-coupled 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-induced 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
Studies on TCR involvement in the repair of MMS-induced lesions have included (i) an analysis of Arg+ revertants, and (ii) examination of plasmid DNA isolated from MMS-treated and transiently starved bacteria 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). Recent studies revealed a significantly slower and completely absent MFD effect in, respectively, AB1157mfd and double alkB mfd mutants. It has been 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 damage to DNA and induction of SOS response. These results again have confirmed the strong mutagenic effects of 1meA/3meC lesions (Wrzesiński et al., 2010).
Interestingly, in a dnaQ mutant no TCR 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 SOS regulon (Grzesiuk & Janion, 1996).
|gln-tRNACAA - tRNA anticodon for glutamine reading 5'CAA3' codon in mRNA|
gln-tRNAUAA - tRNA anticodon reading
|gln-tRNAUAG - tRNA anticodon reading|
gln-tRNAUAA - tRNA anticodon reading
|lys-tRNAAAA - tRNA anticodon for lysine|
reading 5'AAA3' codon in mRNA
lys-tRNAUAA - tRNA anticodon reading
|→||No changes in tRNA encoding genes|
|mutations leading to any sense nucleotide triplet or UAG nonsense codon recognized by |
2.3. The trpE65 → Trp+ and tyrA14 → Tyr+ reversion systems in E. coli B/r derivatives
It is thought that E. coli B is the clonal descendant of a Bacillus coli strain used by Felix d’Herelle from the Pasteur Institute in Paris, in his studies performed on bacteriophages almost 100 years ago. B. coli was isolated from human feces as a normal commensal of the human gut. B. coli was renamed to E.coli strain B and published by Delbrück and Luria in 1942 (Delbrück & Luria in 1942). The history of E.coli B was excellently presented by Daegelen and co-workers (Daegelen et al., 2009). E. coli B/r (B resistant to radiation) is one of the mutants obtained from E. coli B after irradiation with UV light by Evelyn Witkin in 1942 (Witkin,1946). E. coli B was found to be very sensitive to UV irrradiation due to La protease (Lon protein, product of the lon gene) deficiency. E. coli B/r strain owns its UV-resistance to sulA mutation (Studier et al., 2009). SulA protein is synthesized in bacterial cell during the SOS response induction and is a substrate for the Lon protease (Goldberg et al., 1994).
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 to E.coli K12 AB1157 mutation detection systems. 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 any 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 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 and Palmer, 1977; Bockrath et al., 1987). Both systems have been used in MFD studies (Bridges et al., 1967; George & Witkin, 1974; Bockrath and Palmer, 1977; Bockrath et al., 1987).
Besides E. coli WP2 strain, its derivatives are widely used: WP2 carrying pKM101 plasmid from S.typhimurium, WP2 uvrA mutant and WP2 uvrA bearing pKM101. E. coli WP2 and its derivatives are recommended to be used in conjunction with Ames S.typhimurium tester strains to screen various compounds for mutagenic activity (Mortelmans & Riccio, 2000), (see chapter 2.8).
2.4. Lac+ reversion system for determination of base substitutions and frameshift mutations
A commonly used and convenient E. coli K-12 lacZ→Lac+ reversion system allows rapid detection of specificity of mutation. The -galactosidase encoding lacZ gene is a 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 11 mutants (E.coli K12 CC101-111 strains) with a lacZ deletion in the chromosome and F episome with cloned lacZ gene bearing defined mutations (six base substitutions in CC101-106 strains, and five frame shift mutations in CC107-111 strains) have been constructed (Coupples and Miller, 1989; Coupples et al., 1990)
Glutamine at 461 position is essential for -galactosidase activity. In CC101-CC106 strains coding position 461 was changed. Reversion to the Lac+ phenotype is due to a specific base substitution at 461 position restoring the glutamic acid codon. CC107-CC111 carry mutations in the lacZ gene that revert to Lac+ via specific frameshifts. The altered sequences contain monotonous runs of each of the four bases or a run of –G-C- sequences on one strand. Addition or loss of a single base pair or loss of –G-C- sequence lead to reversion of the lacZ mutation. In the case of CC101-CC111 strains the marker is episomal, in contrast to e.g. the AB1157 strain where the marker is situated on the chromosome. Figure 3 presents the idea of the CC101-CC106 and CC107-CC111 strains construction. The lacZ→Lac+ reversion system in E.coli K12 CC101-111 strains is very handy and used all over the world for studying specificity of mutations in genes under investigation.
Fijałkowska and Shaaper with colleagues constructed a series of lacZ strains allowing studies on replication fidelity based on analysis of frequency of Lac+ revertans. The entire lacIZYA operon from Fpro lac plasmid of Coupples and Miller’s strains containing specific lacZ mutation has been inserted into the chromosome of the E. coli MC4100 (lac–) in two possible orientations with regard to the chromosomal replication origin oriC. This system enables investigation of frequencies of base pair substitutions and frame-shift mutations. It has been used to show that during chromosomal DNA replication in E. coli two DNA strands, the leading and the lagging, are replicated with different accuracy in w.t. as well as in various mutants in genes involved in replication or DNA repair (Fijałkowska et al., 1998; Maliszewska-Tkaczyk et al., 2000; Gaweł et al., 2002).
2.5. Forward mutation system with the use of E. coli lacI strain
A forward mutational system, in contrary to reversion systems, monitors the mutation of a wild type gene. The E.coli lacI nonsense system described by Miller and Coulondre (Coulondre & Miller, 1977; Miller, 1983) is a forward system playing an important role in the examination of specificity of numerous mutagens. The lacI gene encodes the repressor of lac operon required to metabolize lactose. The base of lacI system is the analysis of nonsense mutations in the lacI gene (present on an F’ episome). The system also involves several techniques to identify each nonsense mutation. There are over 80 sites within lacI gene where a nonsense mutation can arise by a single base change. Nonsense mutations constitute 20-30% of all mutations induced by many mutagens. Since the lacI gene encodes the repressor of the lac operon, E.coli lacI – cells express the operon constitutively. In this way lacI mutants can be selected on the plates containing phenyl--galactosidase (a lactose analog) as the only source of carbon. These mutants can metabolize the analog but cannot induce the operon. Further mutant analysis involves ability to be suppressed by various tRNA suppressors and subsequent genetic analyses.
The distribution of lacI mutations can be arranged according to base substitution generated by each nonsense mutation, creating a map of mutational hot and cold spots, for places where the number of mutations exceeds or is smaller, respectively, in comparison to other sites. Created map of mutational spectra is characteristic for different mutagens. The lacI nonsense system shows limitations of detecting only base substitution mutations and not detecting AT→GC transitions. Nevertheless, it became possible to determine the nature of the lacI mutations directly by DNA sequencing.
2.6. The trpA → Trp+ reversion system in E.coli K12
An important approach to determine mutagen specificity based on reversion of an auxotrophic trpA mutation to the Trp+ phenotype in E. coli K12 was developed by Yanofsky and co-workers (Berger et al., 1968). The trpA gene is a part of the trp operon and codes for the tryptophan synthetase α chain in E.coli. There is a set of the trpA alleles that enable to monitor all possible base substitutions (trp88, trp46, trp23, trp3, trp223, trp58, trp78, trp11, trp446) and frame shifts (trpE9777, trpA21, trpA540, trpA9813 alleles) and allow studying mutagen specificity. The Trp+ revertants are divided into classes based upon colony size and two physiological tests: 5-methyl tryptophan (5-MT) sensitivity and indole glycerol phosphate (IGP) accumulation. Moreover, full Trp+ revertants (FR) and partial Trp+ revertants (PR) are distinguished. The PR group is divided into 3 more classes: PRI, PRII, PRIII. To enhance the frequiences of spontaneous and induced Trp+ revertants pKM101 plasmid from S. typhimurium (described in chapter 2.8) was introduced to E. coli trp
|AT → TA|
|GC → CG|
AT → TA
|AT → CG|
GC → CG
CG → AT
|AT → GC|
AT → CG
AT → TA
|AT → GC|
AT → CG
GC → AT
|AT → CG|
|AT → CG|
|AT → GC|
AT → CG
2.7. Adaptive mutations
Adaptive mutations (also called “directed”, “stationary phase” or “starvation associated”) are a special kind of spontaneous mutations that occur in non-dividing or slowly-growing stationary-phase cells. Mutations of this type are detectable after exposure to a non-lethal selection and allow growth under these conditions.
As a tool for studying stationary phase mutations,
The systems searching for stationary phase mutations operating on bacterial chromosomal loci use reversion to prototrophy of auxotrophic
In bacteria there is no single mechanism for the generation of stationary-phase mutations. Under starvation conditions mutations can arise as a result of oxidative and other DNA damage, errors occurring during DNA replication, defects or inefficiency of DNA repair systems but also DNA repair synthesis by itself may be a source of mutagenesis under conditions restricted for growth.
2.8. The Ames test with the use of Salmonella strains
The Ames II assay is a liquid microtiter modification of the Ames test. It involves new set of
|STN, N4AC||A:T → G:C|
|STN, MMS||T:A → A:T|
|STN, ANG/UVA||T:A → G:C|
|NQNO, MNNG||G:C → A:T|
|NQNO, MMS||C:G → A:T|
|NQNO, 5azaC||C:G → G:C|
As mentioned in chapter 2.3,
2.9. Test system to study mutations in Pseudomonas putida
There is only a limited number of test systems that allow studying mutagenic processes in
Bacillus subtilisas a model for mutation detection
2.11. Resistance of bacteria to antibiotics
Antibiotic resistance to streptomycin, rifampicin or nalidixic acid is often used for determination of spontaneous and induced mutagenesis in bacteria. It is a universal, rapid and simple test used in many species. The frequency of an antibiotic resistant bacteria usually is determined on plates supplemented with respective antibiotic. Rifampicin-, streptomycin- and nalidixic acid-resistant mutants arise due to spontaneous or induced mutations in chromosomal DNA. The mechanisms leading to rifampicin resistance was described in chapter 2.9. Resistance to streptomycin involves mutational changes in the 30S subunit of the ribosome, whereas resistance to nalidixic acid results from point mutations in structural genes encoding gyrase (topoisomerase II) subunit A (
Resistance to other agents such as mentioned 8-azaguanine (see chapter 2.8) or phages, e.g. T1 phages, has been also used in mutagenesis tests, particularly in the 20th century studies. Resistance to T1 is due to
Living organisms are continuously exposed to damaging agents both from the environment and from endogenous metabolic processes, whose action results in modification of proteins, lipids, carbohydrates and nucleic acids. The knowledge on DNA modifications leading to mutations is critical to our understanding of how and why the genome is affected during the lifespan of the organism, and how the DNA repair systems efficiently work
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