Structure and function of dnaQ and mutD mutators of Escherichia coli

Summary The nucleotide sequences of the recessivednaQ49 and the dominantmutD5 mutator were determined. ThednaQ49 mutator has a single base substitution in thednaQ gene, thus causing one amino acid change,₉₆Val (GTG)→ Gly (GGG), in the DnaQ protein (ε subunit of DNA polymerase III holoenzyme). ThemutD5 mutator possesses two base substitutions in the same gene, resulting in two amino acid changes,⁷³Leu (TTG)→Trp (TGG) and¹⁶⁴Ala (GCA)→Val (GTA), which were designated themutD52 andmutD51 mutations, respectively. Construction of chimaeric genes carrying one or two of these mutations revealed: (1) eithermutD51 ormutD52 alone causes the dominant mutator phenotype when present in a multi-copy plasmid; (2)mutD51, but notmutD52, exerts the dominant mutator phenotype when present in a low-copy plasmid; (3) the dominantmutD51 mutator activity is suppressed by thednaQ49 mutation when both mutations are present in the same gene. Based on these findings, we devised a model for the action of these mutators.     

Escherichia coli mutator (Delta)polA is defective in base mismatch correction: the nature of in vivo DNA replication errors

We constructed a set of Escherichia coli strains containing deletions in genes encoding three SOS polymerases, and defective in MutS and DNA polymerase I (PolI) mismatch repair, and estimated the rate and specificity of spontaneous endogenous tonB(+)-->tonB- mutations. The rate and specificity of mutations in strains proficient or deficient in three SOS polymerases was compared and found that there was no contribution of SOS polymerases to the chromosomal tonB mutations. MutS-deficient strains displayed elevated spontaneous mutation rates, consisting of dominantly minus frameshifts and transitions. Minus frameshifts are dominated by warm spots at run-bases. Among 57 transitions (both G:C-->A:T and A:T-->G:C), 35 occurred at two hotspot sites. PolI-deficient strains possessed an increased rate of deletions and frameshifts, because of a deficiency in postreplicative deletion and frameshift mismatch corrections. Frameshifts in PolI-deficient strains occurred within the entire tonB gene at non-run and run sequences. MutS and PolI double deficiency indicated a synergistic increase in the rate of deletions, frameshifts and transitions. In this case, mutS-specific hotspots for frameshifts and transitions disappeared. The results suggested that, unlike the case previously known pertaining to postreplicative MutS mismatch repair for frameshifts and transitions and PolI mismatch repair for frameshifts and deletions, PolI can recognize and correct transition mismatches. Possible mechanisms for distinct MutS and PolI mismatch repair are discussed. A strain containing deficiencies in three SOS polymerases, MutS mismatch repair and PolI mismatch repair was also constructed. The spectrum of spontaneous mutations in this strain is considered to represent the spectrum of in vivo DNA polymerase III replication errors. The mutation rate of this strain was 219x10(-8), about a 100-fold increase relative to the wild-type strain. Uncorrected polymerase III replication errors were predominantly frameshifts and base substitutions followed by deletions.     

Temperature-dependent mutational specificity of an Escherichia coli mutator, dnaQ49, defective in 3'----5' exonuclease (proofreading) activity

Escherichia coli strains carrying the temperature-dependent dnaQ49 allele are strong mutators at 37 degrees C. Since the dnaQ49 gene encodes the epsilon subunit of DNA polymerase III, it is thought that the large number of errors results in part from impaired proofreading activity during DNA replication. We have examined dnaQ49-induced reversion patterns of defined trpA alleles to determine the kinds of errors produced by dnaQ49 at 30 degrees C and 37 degrees C. We found that at 37 degrees C dnaQ49 produced all types of base-pair substitutions in addition to frameshifts with transitions generally occurring more frequently than transversions. This generalized mutator activity is very similar to that displayed in rich medium by mutD5, another mutator allele at the dnaQ locus. However, when dnaQ49 strains were cultured at 30 degrees C, not only were reversion frequencies much lower than at 37 degrees C, but in addition, the spectrum was altered. Transversions became proportionally more prevalent in the reversion spectra at the lower temperature. We suggest the possibility that at 37 degrees C dnaQ49 results in defective proofreading and methyl-directed postreplicative mismatch repair, while at 30 degrees C mismatch repair is fully and proofreading partially restored.     

Mutational specificity of ethyl methanesulfonate in excision-repair-proficient and -deficient strains of Drosophila melanogaster

Summary The vermilion gene was used as a target to determine the mutational specificity of ethyl methanesulfonate (EMS) in germ cells of Drosophila melanogaster. To study the impact of DNA repair on the type of mutations induced, both excision-repair-proficient (exr ⁺) and excision-repair-deficient (exr ^–) strains were used for the isolation of mutant flies. In all, 28 mutants from the exr ⁺ strain and 24 from the exr ^– strain, were characterized by sequence analysis. In two mutants obtained from the exr ⁺ strain, small deletions were observed. All other mutations were caused by single base-pair changes. In two mutants double base-pair substitutions had occurred. Of the mutations induced in the exr ⁺ strain, 22 (76%) were GCAT transitions, 3 (10%) ATTA transversions, 2 (6%) GCTA transversions and 2 (6%) were deletions. As in other systems, the mutation spectrum of EMS in Drosophila is dominated by GCAT transitions. Of the mutations in an exr ^– background, 12 (48%) were GCAT transitions, 7 (28%) ATTA transversions, 5 (20%) GCTA transversions and 1 (4%) was a ATGC transition. The significant increase in the contribution of transversion mutations obtained in the absence of an active maternal excision-repair mechanism, clearly indicates efficient repair of N-alkyl adducts (7-ethyl guanine and 3-ethyl adenine) by the excision-repair system in Drosophila germ cells.     

Mutagenic DNA repair in Escherichia coli. XIV. Influence of two DNA polymerase III mutator alleles on spontaneous and UV mutagenesis

Summary We introduced the dnaE486 and polC74 mutations (which are associated with decreased DNA polymerase III replication fidelity) into excision defective Escherichia coli strains with varying SOS responses. These mutations increased the UV-induced frequency of base pair substitution mutations in all strains tested, except recA430 and umuC122 derivatives. This UV mutator effect therefore requires expression of the SOS error-prone repair system. In recA441 lexA51 strains where the SOS system is constitutively expressed, the UV mutator effect of the dnaE alleles was similar in relative terms (though greater in absolute terms). Since these dnaE alleles decrease rather than increase survival after UV it is argued that they promote a burst of untargeted mutations close to UV photoproducts (hitch-hiking mutations) rather than increase the number of translesion synthesis events. The fact that there was no UV mutagenesis in dnaE486 umuC122 or polC74 umuC122 strains indicates that infidelity associated with these dnaE alleles did not of itself enable translesion synthesis to occur. The spontaneous mutator effect conferred by dnaE486 and polC74 was not affected by umuC122 or recA430 indicating that it is not dependent upon error-prone repair ability. In recA441 lexA51 bacteria, where SOS error-prone repair is constitutively induced, the mutator effect of dnaE486 was greater and was largely blocked by umuC122. It is suggested that spontaneously occurring cryptic lesions that are themselves unable to induce the SOS system are subject to translesion synthesis under these conditions and trigger a burst of hitch-hiking mutations that are therefore effectively umuC dependent.     

Nature of the SOS mutator activity: genetic characterization of untargeted mutagenesis in Escherichia coli

Summary In Escherichia coli, induction of the SOS functions by UV irradiation or by mutation in the recA gene promotes an SOS mutator activity which generates mutations in undamaged DNA. Activation of RecA protein by the recA730 mutation increases the level of spontaneous mutation in the bacterial DNA. The number of recA730-induced mutations is greatly increased in mismatch repair deficient strains in which replication errors are not corrected. This suggests that the majority of recA730-induced mutations (90%) arise through correctable, i.e. non-targeted, replication errors. This recA730 mutator effect is suppressed by a mutation in the umuC gene. We also found that dam recA730 double mutants are unstable, segregating clones that have lost the dam or the recA mutations or that have acquired a new mutation, probably in one of the genes involved in mismatch repair. We suggest that the genetic instability of the dam recA730 mutants is provoked by the high level of replication errors induced by the recA730 mutation, generating killing by coincident mismatch repair on the two unmethylated DNA strands. The recA730 mutation increases spontaneous mutagenesis of phage poorly. UV irradiation of recA730 host bacteria increases phage untargeted mutagenesis to the level observed in UV-irradiated recA ⁺ strains. This UV-induced mutator effect in recA730 mutants is not suppressed by a umuC mutation. Therefore UV and the recA730 mutation seem to induce different SOS mutator activities, both generating untargeted mutations.     

A mutator nuclear gene inducing a wide spectrum of cytoplasmically inherited chlorophyll deficiences in barley

Summary Some striped plants were observed in plots of a long-grain mutant barley grown at a field nursery. All of the plants of these plots, which were naturally self pollinated, were individually harvested, and most of their progenies (92.5%) segregated seedlings carrying chlorophyll deficiencies (CD) as determined by greenhouse analysis. The majority of the mutant seedlings (84.3%) showed a pattern of longitudinal chlorophyll sectors. The spectrum of CD was wide among the solid mutant seedlings and consisted of three main types (albina, viridis and discontinuous). In association with some CD types morphological changes were frequently observed. Non-CD-associated morphological changes and diminished seed-set were scarce and, so far, none of them has proved to be inherited. Analysis of CD in reciprocal crosses and backcrosses proved that while CD were transmitted cytoplasmically their induction was controlled by a single nuclear mutator gene, active when homozygous. In addition once the CD were induced, they were expressed independently of the nuclear constitution. The results suggest that the mutator gene induces diverse mutational events on chloroplast (cp) DNA. In barley, as in other monocots, nuclear genes which are inductors of cytoplasmic genetic changes have been reported. However, all of them produced a narrower spectrum of CD and had a more rapid sorting-out of the cytoplasmic mutants than what we observed. On this basis a distinction between chloroplast and mitochondrial (mt) mutator genes is proposed. Accordingly, the chloroplast mutator here described would be the first one reported for monocots. Increased knowledge on this subject can play a fundamental role in elucidating organelle heredity and its interactions with the nuclear genome. Moreover, this material could be a valuable source of variability of the otherwise conservative genetic information encoded in the chloroplast.Paper GEN 792, Institute of Genetics, CICA, INTA, Castelar     

Tcl transposition and mutator activity in a Bristol strain of Caenorhabditis elegans

Summary In most strains of Caenorhabditis elegans with a low copy number of Tc1 transposable elements, germline transposition is rare or undetectable. We have observed low-level Tel transposition in the genome of the C. elegans var. Bristol strain KR579 (unc-13[e51]) resulting in an increase in Tc1 copy number and subsequent mutator activity. Examination of genomic blots from KR579 and KR579derived strains revealed that more Tc1-hybridizing bands were present than in other Bristol strains. A novel Tc1-hybridizing fragment was cloned from a KR579-derived strain. Unique sequence DNA flanking the Tc1 element identified a 1.6 kb restriction fragment length difference between the KR579 and N2 strains consistent with a Tc1 insertion at a new genomic site. The site of insertion of this Tel was sequenced and is similar to the published Tel insertion site consensus sequence. Several isolates of KR579 were established and maintained on plates for a period of 3 years in order to determine if Tc1 copy number would continue to increase. In one isolate, KR1787, a further increase in Tc1 copy number was observed. Examination of the KR1787 strain has shown that it also exhibits mutator activity as assayed by the spontaneous mutation frequency at the unc-22 (twitcher) locus. The KR579 strain differs from most low copy number strains in that it exhibits low-level transposition which has developed into mutator activity.     

Increased expression of the Escherichia coli umuDC operon restores SOS mutagenesis in lexA41 cells

Summary The lexA41 allele of Escherichia coli encodes a semidefective mutant repressor that is also resistant to RecA facilitated cleavage. Cells harboring the lexA41 allele were found previously to repress only a subset of operons in the SOS regulon. lexA41 cells cannot promote SOS mutagenesis, presumably because one or more operons required for mutagenesis are repressed by this mutant repressor. Using the lac regulatory system to increase the expression of the umuDC operon, we were able to restore mutagenesis in the lexA41 mutant. We conclude that the products of the umuDC operon appear to be uniquely limiting in this mutant.     

Mutational spectrum induced in Saccharomyces cerevisiae by the carcinogen N-2-acetylaminofluorene

The spectrum of mutations induced by the carcinogen N-2-acetylaminofluorene (AAF) was analysed in Saccharomyces cerevisiae using a forward mutation assay, namely the inactivation of the URA3 gene. The URA3 gene, carried on a yeast/bacterial shuttle vector, was randomly modified in vitro using N-acetoxy-N-2-acetylaminofluorene (N-AcO-AAF) as a model reactive metabolite of the carcinogen AAF. The binding spectrum of AAF to the URA3 gene was determined and found to be essentially random, as all guanine residues reacted about equally well with N-AcO-AAF. Independent Ura^– mutants were selected in vivo after transformation of the modified plasmid into a ura3 yeast strain. Plasmid survival decreased as a function of AAF modification, leading to one lethal hit (37% relative survival) for an average of 50 AAF adducts per plasmid molecule. At this level of modification the mutation frequency was equal to 70 × 10^–4, i.e. 50-fold above the background mutation frequency. UV irradiation of the yeast cells did not further stimulate the mutagenic response, indicating the lack of an SOS-like mutagenic response in yeast. Sequence analysis of the URA3 mutants revealed 48% frameshifts, 44% base substitutions and 8 % complex events. While most base substitutions (74%) were found to be targeted at G residues where AAF is known to form covalent C8 adducts, frameshift mutations were observed at GC base pairs in only 24% of cases. Indeed, more than 60% of frameshift events occurred at sequences such as 5-(A/T)_nG-3 where a short (n = 2 or 3) monotonous run of As or Ts is located on the 5' side of a guanine residue. We refer to these mutations as semi-targeted events and present a potential mechanism that explains their occurrence.     

UV irradiation inhibits initiation of DNA replication from oriC in Escherichia coli

Summary Irradiation of Escherichia coli with UV light causes a transient inhibition of DNA replication. This effect is generally thought to be accounted for by blockage of the elongation of DNA replication by UV-induced lesions in the DNA (a cis effect). However, by introducing an unirradiated E. coli origin (oriC)-dependent replicon into UV-irradiated cells, we have been able to show that the environment of a UV-irradiated cell inhibits initiation of replication from oriC on a dimer-free replicon. We therefore conclude that UV-irradiation of E. coli leads to a trans-acting inhibition of initiation of replication. The inhibition is transient and does not appear to be an SOS function.     

The Escherichia coli dam gene is expressed as a distal gene of a new operon

Summary DNA containing the Escherichia coli dam gene and sequences upstream from this gene were cloned from the Clarke-Carbon plasmids pLC29-47 and pLC13-42. Promoter activity was localized using pKO expression vectors and galactokinase assays to two regions, one 1650–2100 bp and the other beyon 2400 bp upstream of the dam gene. No promoter activity was detected immediately in front of this gene; plasmid pDam118, from which the nucleotide sequence of the dam gene was determined, is shown to contain the pBR322 promoter for the primer RNA from the pBR322 rep region present on a 76 bp Sau3A fragment inserted upstream of the dam gene in the correct orientation for dam expression. The nucleotide sequence upstream of dam has been determined. An open reading frame (ORF) is present between the nearest promoter region and the dam gene. Codon usage and base frequency analysis indicate that this is expressed as a protein of predicted size 46 kDa. A protein of size close to 46 kDa is expressed from this region, detected using minicell analysis. No function has been determined for this protein, and no significant homology exist between it and sequences in the PIR protein or GenBank DNA databases. This unidentified reading frame (URF) is termed urf-74.3, since it is an URF located at 74.3 min on the E. coli chromosome. Sequence comparisons between the regions upstream of urf-74.3 and the aroB gene show that the aroB gene is located immediately upstream of urf-74.3, and that the promoter activity nearest to dam is found within the aroB structural gene. This activity is relatively weak (about 15% of that of the E. coli gal operon promoter). The promoter activity detected beyond 2400 bp upstream of dam is likely to be that of the aroB gene, and is 3 to 4 times stronger than that found within the aroB gene. Three potential DnaA binding sites, each with homology of 8 of 9 bp, are present, two in the aroB promoter region and one just upstream of the dam gene. Expression through the site adjacent to the dam gene is enhanced 2-to 4-fold in dnaA mutants at 38°C. Restriction site comparisons map these regions precisely on the Clarke-Carbon plasmids pLC13-42 and pLC29-47, and show that the E. coli ponA (mrcA) gene resides about 6 kb upstream of aroB.     

Mutational specificity of a bacteriophage T4 DNA polymerase mutant, mel88

Summary Several bacteriophage T4 DNA polymerase mutants have been shown to increase the frequency of spontaneous mutations (Speyer et al. 1966; Freese and Freese 1967; de Vries et al. 1972; Reha-Krantz et al. 1986). In order to determine the molecular basis of the mutator phenotype, it is necessary to characterize the types of mutations produced by the mutator DNA polymerases. We show here that at least one DNA polymerase mutator mutant, mel88, induces an increased number of base substitution mutations compared with wild-type.     

DNA polymerase V-dependent mutator activity in an SOS-induced Escherichia coli strain with a temperature-sensitive DNA polymerase III.

The temperature-sensitive DNA polymerase III (Pol III) encoded by the dnaE486 allele confers a spontaneous mutator activity in SOS-induced bacteria that is largely dependent upon DNA polymerase V (Pol V), encoded by umuD, C. This mutator activity is influenced by the defective proof-reading sub-unit of Pol III encoded by the dnaQ905 (mutD5) allele arguing that Pol V is most likely fixing mutations arising from mismatched primer termini produced by Pol III(486). The size of the dnaQ effect is, however, modest leaving open the possibility that Pol V may be responsible for some of the mutator effect by engaging in bursts of processive activity.     

Host controlled plasmid replication: Escherichia coli minichromosomes

Escherichia coli minichromosomes are plasmids replicating exclusively from a cloned copy of oriC, the chromosomal origin of replication. They are therefore subject to the same types of replication control as imposed on the chromosome. Unlike natural plasmid replicons, minichromosomes do not adjust their replication rate to the cellular copy number and they do not contain information for active partitioning at cell division. Analysis of mutant strains where minichromosomes cannot be established suggest that their mere existence is dependent on the factors that ensure timely once per cell cycle initiation of replication. These observations indicate that replication initiation in E. coli is normally controlled in such a way that all copies of oriC contained within the cell, chromosomal and minichromosomal, are initiated within a fairly short time interval of the cell cycle. Furthermore, both replication and segregation of the bacterial chromosome seem to be controlled by sequences outside the origin itself.     

Sequence analysis and phenotypes of five temperature sensitive mutator alleles of dnaE, encoding modified alpha-catalytic subunits of Escherichia coli DNA polymerase III holoenzyme.

In the 1970s, several thermosensitive alleles of dnaE (encoding the alpha-catalytic subunit of pol III) were isolated. Genetic characterization of these dnaE mutants revealed that some are mutator alleles at permissive temperature. We have determined the nucleotide changes of five such temperature sensitive mutator alleles (dnaE9, dnaE74, dnaE486, dnaE511, and dnaE1026) and find that most are single missense mutations. The exception is dnaE1026 which is a compound allele consisting of multiple missense mutations. When the previously characterized mutator alleles were moved into a lexA51(Def) recA730 strain, dnaE486, dnaE1026 and dnaE74 conferred a modest approximately two-six-fold increase in spontaneous mutagenesis when grown at the permissive temperature of 28 degrees C, while dnaE9 and dnaE511 actually resulted in a slight decrease in spontaneous mutagenesis. In isogenic DeltaumuDC derivatives, the level of spontaneous mutagenesis dropped significantly, although in each case, the overall mutator effect conferred by the dnaE allele was relatively larger, with all five dnaE alleles conferring an increased spontaneous mutation rate approximately 5-22-fold over the isogenic dnaE+ DeltaumuDC strain. Interestingly, the temperature sensitivity conferred by each allele varied considerably in the lexA51(Def) recA730 background and in many cases, this phenotype was dependent upon the presence of functional pol V (UmuD'2C). Our data suggest that pol V can compete effectively with the impaired alpha-subunit for a 3' primer terminus and as a result, a large proportion of the phenotypic effects observed with strains carrying missense temperature sensitive mutations in dnaE can, in fact, be attributed to the actions of pol V rather than pol III.     

Mutations in the DnaA binding sites of the replication origin of Escherichia coli.

Summary Mutations (base changes) were introduced into the four DnaA binding sites (DnaA boxes) of theEscherichia coli replication origin,oriC. Mutations in a single DnaA box did not impair the ability of these origins to replicate in vivo and in vitro. A combination of mutations in two DnaA boxes, R1 and R4, resulted in slower growth of theoriC plasmid-bearing host cells. DnaA protein interaction with mutant and wild-type DnaA boxes was analyzed by DNase I footprinting. Binding of DnaA protein to a mutated DnaA box R1 was not affected by a mutation in DnaA box R4 and vice versa. Mutations in DnaA boxes R1 and R4 did not modify the ability of the DnaA protein to bind to other DnaA boxes inoriC.