umuC Gene Research Articles

Expression of the SOS system in Escherichia coli growing under nitrate respiration conditions

Induction of several SOS functions by mitomycin C, bleomycin or thermal treatment of a recA441 mutant growing under nitrate respiration conditions was studied in Escherichia coli. Mitomycin C caused inhibition of cell division, induction of prophages and expression of umuC gene but like in aerobically growing cells, it did not trigger the cessation of cell respiration. On the contrary, both recA+ and recA441 cultures either treated with bleomycin or incubated at 42 degrees C failed to induce any of the different SOS functions cited above. Furthermore, after bleomycin addition or thermal treatment both recA+ and recA441 cultures did not present any variation in the cellular ATP level, contrary to what happens under aerobic growth. The blocking of the expression of some SOS functions under nitrate respiration conditions is not an irreversible process because cells incubated under these anaerobic conditions were able to induce the SOS system when changed to an aerobic medium 30 min after the SOS-inducing treatment had been applied.

Mutagenic DNA repair in Escherichia coli. XII. Ultraviolet mutagenesis in excision-proficient umuC and lexA (ind-) bacteria as revealed by delayed photoreversal.

Streptomycin-resistant mutations are induced in excision-proficient umuC122::Tn5 bacteria given delayed photoreversal after u.v. light. The mutations occur after much earlier photoreversal than is found with the similar induction of tryptophan-independent mutations in excision-deficient umuC bacteria and the process is not markedly influenced by the presence of Casamino acids. The yield of mutants is between 25 and 40% of that found immediately after u.v. irradiation of isogenic umu+ bacteria. Mutagenesis is not inhibited by the presence of chloramphenicol after u.v. and before photoreversal when the bacteria have also been pretreated with chloramphenicol. Loss of photoreversibility of streptomycin-resistant mutations in umu+ excision-proficient bacteria also occurs during a similar period after u.v. and is similarly unaffected by Casamino acids and chloramphenicol. The results are interpreted on a 2-step model of error-prone repair in which a misincorporation step is followed by a lesion bypass step which requires induced levels of umuC gene product, the latter step being unnecessary when the pyrimidine dimer is removed by photoreversal after the misincorporation has taken place. Differences from the same phenomenon in excision-deficient bacteria are accounted for by the fact that in excision-proficient bacteria the mutation process is initiated by excision repair rather than during normal chromosomal replication. It is suggested that loss of photoreversibility in excision-proficient umu+ bacteria may reflect the misincorporation step only, thus explaining the apparent non-inducible nature of loss of photoreversibility previously reported. The bypass step alone requires induced levels of umuD,C gene products as is evidenced by the u.v.-induction of mutations in lexA102(ind-) bacteria after delayed photoreversal.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of an umuC mutation on phage λ induction

A possible role of the umuC gene product in the induction of the SOS responses was examined. We compared the expression of a genetic fusion, in which gene lacZ, encoding beta-galactosidase in Escherichia coli is under the direct control of the cI repressor from prophage lambda, in a umuC+ strain and in an otherwise isogenic umuC- mutant. We found that two times higher UV doses were required to obtain a similar induction in the umuC+ strain as in the umuC mutant. In addition we showed that, at the same UV dose after a lag period, the specific activity of beta-galactosidase increased more rapidly in the umuC mutant. We suggest that the wild-type umuC gene product participates in the processing of the SOS inducing structures caused by UV irradiation or prevents formation of some of them. This is compatible with a role of the UmuC protein in DNA synthesis past a replication block.

Weigle reactivation and mutagenesis of bacteriophage lambda in lexA(Def) mutants of E. coli K12.

The SOS response in UV-irradiated bacteria enhances the survival and mutagenesis of infecting damaged bacteriophage lambda. In a lexA(Def) strain, SOS bacterial genes are fully derepressed by an inactivating mutation in the LexA repressor gene. We tested several lexA(Def) derivative strains for their capacity to constitutively promote high survival and mutagenesis of irradiated lambda. We showed that UV irradiation of the lexA(Def) host bacteria is still necessary for optimal efficiency of both these SOS functions, which are dependent on the umuC gene product and an activated form of RecA protein.

Cellular responses to DNA damage.

For many years, the study of the regulation of the SOS network was complicated by both the complexities of the responses and the interrelationships of the key regulatory elements. However, recently the application of powerful genetic and molecular biological techniques has allowed us to gain a detailed picture of the regulation of this complex network. The network is now known to consist of more than 17 genes, each of which is repressed by the LexA protein. Induction of the genes in the SOS network occurs when the RecA protein becomes activated in response to a signal generated by DNA damage. Two of the genes in this network, umuD and umuC, are absolutely required for mutagenesis by UV and various carcinogens. The umuD and umuC genes have molecular weights of 16,000 and 45,000 daltons, respectively, and are organized in an operon repressed by LexA. The mutagenesis-enhancing plasmid pKM101 carries two genes mucA and mucB, which are analogs of the umuD and umuC genes, respectively.

Evaluation of the new system ( umu-test) for the detection of environmental mutagens and carcinogens

The umu operon in Escherichia coli is responsible for chemical and radiation mutagenesis, and the expression of the operon itself is inducible by these DNA-damaging agents. The principle of the umu-test is based on the ability of the DNA-damaging agents, most of which are potential carcinogens, to induce the umu operon. A plasmid (pSK1002) carrying a fused gene umuC′-′lacZ was introduced into Salmonella typhimurium TA1535. The strain TA1535/pSK1002 enabled us to monitor the levels of umu operon expression by measuring the β-galactosidase activity in the cells produced by the fusion gene. Using this strain, a simple, inexpensive, and sensitive system, the umu-test, for the screening of environmental mutagens and carcinogens was developed. 38 chemicals with different structures and modes of action, including 31 known animal carcinogens, were examined by the test to evaluate the system. The threshold sensitivity of the umu-test was approximately equal to that of the Ames test for chemicals genotoxic in both tests. By the umu-test, using the single tester strain, we detect many types of DNA-damaging agents for which the Ames test requires several tester strains. Furthermore, the umu-test provides a potential practical advantage for the screening of various environmental samples containing amino acids and nutrients such as urine, serum and foods.

Photoreactivation of Escherichia coli reverses umuC induction by UV light.

UV mutagenesis in Escherichia coli depends on the presence of a premutagenic lesion in DNA and on the induction of the umuCD gene product as part of the SOS response. Using operon fusions between the E. coli lacZ gene and the SOS genes umuC, uvrB, and dinD, we have affirmed the expected role of the cyclobutane pyrimidine dimer in inducing SOS gene transcription. In addition, we found that photoreactivation of cyclobutane pyrimidine dimers reversed umuC induction to the same extent as it reversed mutagenesis. Therefore, the photoreactivability of UV mutagenesis does not itself identify the cyclobutane pyrimidine dimer as the UV mutagenic lesion in E. coli.

Effect of deficiency in excision repair and umuC function on the mutagenicity with ethylene oxide in the lacI gene of E. coli

The influence of uvrB and umuC genes on the induction of lacI − mutants and nonsense mutants by ethylene oxide (EtO) in the lacI gene of E. coli was studied. The uvrB mutation was characterized by much higher mutation frequencies. In contrast the umuC mutation does not significantly affect the induction kinetics. Thus mutation by EtO is enhanced by the lack of excision repair but not influenced by error-prone repair.

Structural analysis of the umu operon required for inducible mutagenesis in Escherichia coli.

The nucleotide sequence of a 2.8-kilobase fragment of the Escherichia coli chromosome containing the umuDC genes has been determined. The DNA sequence specifies two open reading frames of 417 and 1266 nucleotides encoding proteins with calculated molecular weights of 15,063 and 47,677, respectively. From these and the previous results of genetic and biochemical studies on the cloned genes, we conclude that the former is the umuD and the latter is the umuC gene. In vitro transcription of the regulatory region of the umu operon revealed that (i) upstream of the coding region there is a promoter-operator complex having the consensus sequence, CTGTATATAAAAACAG, of an SOS box, (ii) transcription of the umu operon begins at an adenine residue in the SOS box, and (iii) the LexA protein binds to the operator region, with an equilibrium dissociation constant (Kd) of 0.2, thereby repressing the transcription of the umuDC genes. These results indicate that the expression of the umu operon is under the coordinated control of the recA lexA gene products.

Mutagenic repair in Escherichia coli: products of the recA gene and of the umuD and umuC genes act at different steps in UV-induced mutagenesis.

When excision-deficient Escherichia coli carrying umuC or umuD alleles were exposed to visible light several hours after ultraviolet irradiation, base-pair-substitution mutations were induced in these normally non-UV-mutable bacteria. It is argued that delayed photoreversal of pyrimidine dimers removes blocks to DNA replication and allows the "survival" and expression of misincorporated bases. A model for UV mutagenesis is proposed with two steps: (i) misincorporation opposite a photoproduct, which can be mediated directly by RecA protein, and (ii) bypass, only the latter process requiring umuD+ and umuC+ alleles. Basal levels of gene products are sufficient for at least some misincorporation events, although induced levels of umuD and umuC gene products are necessary for the bypass step. umuC bacteria containing the recA441 allele showed a greater yield of mutants, and those containing recA430 a reduced yield, following delayed photoreversal. The lexA51 allele (which results in constitutive derepression of RecA protein production) did not significantly alter the yield of mutants but caused them to appear marginally sooner in a recA441 umuC strain. These results emphasize that the nature of the RecA protein and not its concentration is paramount in determining the level of misincorporation. Experiments with recA441 umuC bacteria at 43 degrees C and 30 degrees C suggest that the misincorporation effect is unlikely to be attributable to cleavage of a DNA binding protein such as a repressor or a component of the polymerase complex. Moreover, misincorporation seems to occur without the need for induced synthesis of any other protein under recA control.

Regulation of the SOS response analyzed by RecA protein amplification.

A split UV light dose procedure was used in Escherichia coli to induce an SOS function, RecA protein amplification, which was measured by an immunoradiometric assay. The SOS system was partially induced after the first UV irradiation, and the inducing effects of subsequent identical UV doses were quantified. Variations in the inducing effects of successive UV doses were related to modulations of the SOS signal level during SOS induction. A reduction in the level of SOS signal was found after 20 min in the wild-type strain, hypothesized to result from negative control of repair functions. A few DNA repair mutants were tested by the same procedure; the uvrA, recF, and umuC genes were involved in SOS induction control, but we found differences in their respective kinetics of expression. On the contrary, in a recB mutant, only a slight effect was obtained on this control.

DNA binding and mutation spectra of the carcinogen N-2-aminofluorene in Escherichia coli: A correlation between the conformation of the premutagenic lesion and the mutation specificity

When the chemical carcinogen N-2-acetylaminofluorene binds to DNA in vivo, two major adducts are formed, both at position C-8 of the guanine residue. One of these (the acetylaminofluorene adduct) retains the acetyl group, while the other (the aminofluorene adduct) is the corresponding deacetylated form. Unlike -AAF † † Abbreviations used: AAF, N-2-acetylaminofluorene; -AAF, acetylaminofluorene; -AF, aminofluorene; N-Aco-AAF. N-acetoxy- N-2-acetylaminofluorene; N-OH-AF, N-hydroxy- N-2-aminofluorene; 2-AF, N-2-aminofluorene; u.v., ultraviolet light. adducts, which trigger important structural changes of the DNA secondary structure (either the insertion-denaturation model or the induction of a Z-DNA structure, depending upon the local nucleotide sequence), -AF adducts bind to the C-8 of guanine residues without causing any major conformational change of the B-DNA structure. Well-defined adducts (either -AF or -AAF) can be formed in vitro by reacting DNA with either N-hydroxy- N-2-aminofluorene or N-acetoxy- N-2-acetylaminofluorene. Specific cleavage of the phosphodiester backbone at -AF adducts can be achieved by treating -AF-modified DNA in 1 m-piperidine at 90 °C. This observation led us to construct the spectrum for -AF binding to a defined DNA restriction fragment. It is found that only guanine residues react to form alkali-labile lesions and that the reactivity among the different guanines is similar. In a forward mutation assay, namely the inactivation of the tetracycline resistance gene, we found previously that more than 90% of mutations induced by -AAF adducts are frameshift mutations. Using the same assay, we show here that -AF aducts induce primarily base substitution mutations (85%), mainly of the G to T transversion type. There is therefore a strong correlation between the nature of the carcinogen-induced conformational change of the DNA structure and the corresponding mutation specificity. The -AF-induced base substitution mutations depend upon the umuC gene function(s). The data obtained in our forward mutation assay are compared to the data previously obtained in the histidine reversion assay (Ames test).

A loss of uvA function decreases the induction of the SOS functions recA and umuC by mitomycin C in Escherichia coli

We have studied the levels of recA and umuC protein synthesis in Escherichia coli as a probe for regulatory and mechanistic events involved in mitomycin C mutagenesis. Both RecA and UmuC protein induction were greatly stimulated by mitomycin C in the wild-type strain, reached a peak at about 60 min for the recA gene, and at 90 min for the umuC gene, respectively, and maintained a plateau. The induction was blocked by recA and lexA(Ind −) mutations that conferred no mutagenesis on the cell. Mutation affecting uvrA protein markedly decreased induction of the recA gene as well as the umuC gene by mitomycin C. The results established that UvrA protein is involved in the induction of recA and umuC, and account, at least in part, for the mitomycin C nonmutability of uvrA mutants.

Role of the umuC gene in postreplication repair in UV-irradiated Escherichia coli K-12 uvrB

The role of the umuC gene product in postreplication repair was studied in UV-irradiated Escherichia coli K-12 uvrB cells. A mutation at umuC increased the UV radiation sensitivities of uvrB, uvrB recF, uvrB recB, and uvrB recF recB cells; it also increased the deficiencies in the repair of DNA daughter-strand gaps in these strains, but it did not affect the repair of DNA double-strand breaks that arose from unrepaired DNA daughter-strand gaps. We suggest that the umuC gene product is involved in a minor system for the repair of DNA daughter-strand gaps, possibly the repair of overlapping DNA daughter-strand gaps.

Weigle reactivation of phage lambda in a recA mutant of Escherichia coli: Dependence on the excess amounts of photoreactivating enzyme in the dark

The plating efficiency of ultraviolet-light-irradiated phage λ in the dark is increased when an Escherichia coli recA host, which is transformed with a multicopy plasmid, pKY1, carrying the phr gene of E. coli, is irradiated with 254 nm UV prior to infection (Weigle (W) reactivation). Such W reactivation in lexA3 and umuC strains, with or without pKY1, is almost undetectable. Addition of umu mutations to recA56/pKY1 cells blocks this process, but addition of the lexA3 mutation instead gives high levels of W reactivation. Fusion of the lacZ gene with recA or umuC promoters permitted measurement of the effects of UV radiation on transcription of these SOS operons for various mutated cells, with and without the phr + plasmid. The presence of the recA56 allele totally abolishes UV induction of both recA and umuC gene expression in the cells, both with and without the plasmid. Thus, the observed W reactivation in the recA strain carrying the phr + plasmid requires only the constitutive amount of the Umu proteins, and does not require the lexA + recA +-dependent inducible process. While we observed an increase in the W reactivation of UV-irradiated λ phage in a recA56/pKY1 cell, there is no corresponding mutagenesis of the UV-irradiated phage in UV-irradiated cells. We propose that the E. coli phr gene product facilitates error-free pathways of DNA repair in the absence of photoreactivation. The fact that a recA/pKY1 strain permits almost normal levels of W reactivation, but completely blocks W mutagenesis, leads us further to suggest that the recA gene product, in general, functions in a regulatory manner in W reactivation and in both regulatory and mechanistic ways in W mutagenesis.

Mechanism of transient inhibition of DNA synthesis in ultraviolet-irradiated E. coli: inhibition is independent of recA whilst recovery requires RecA protein itself and an additional, inducible SOS function.

The mechanism of the inhibition and of the recovery of DNA synthesis in E. coli following UV-irradiation was analysed in several mutants defective in repair or in the regulation of the RecA-LexA dependent SOS response. Several lines of evidence indicated that inhibition is not an inducible function and is probably due to the direct effect of lesions in the template blocking replisome movement. Recovery of DNA synthesis after UV was largely unaffected by mutations in the uvrA, recB or umuC genes. Resumption of DNA synthesis does however require protein synthesis and the regulatory action of recA. Experiments with a recA constitutive mutant and recA 200 (temperature sensitive RecA) demonstrated that RecA protein itself is directly required but is not sufficient for recovery of DNA synthesis. We therefore propose that recovery of DNA synthesis depends upon the concerted activity of RecA and the synthesis of an inducible Irr (induced replisome reactivation) factor under RecA control. We suggest that the mechanism of recovery involves the action of Irr and RecA to promote movement of replisomes past non-instructive lesions, uncoupled from polymerisation and/or that Irr and RecA are required to promote re-initiation of a stalled replication complex downstream of a UV-lesion subsequent to such an uncoupling step.

Induction of umuC gene expression by nitrogen dioxide in Salmonella typhimurium

Gaseous nitrogen dioxide (NO 2) was found to induce umuC gene expression in Salmonella typhimurium carrying the umuC-lacZ fusion plasmid. The induction level of the umu operon responsible for inducible mutagenesis was measured by the level of β-galactosidase in the cell, encoded by the fusion gene. NO 2 gas was bubbled into bacterial suspensions at 10, 30 and 90 μl/l for 30 min at a flow rate of 100 ml/min. Expression of the umuC gene varied with the concentration, flow rate and bubbling time of the NO 2 gas. Although NO 2 gas induces SOS functions, mutagenesis due to it was not detectable in Salmonella typhimurium TA100 and TA102. Nitric oxide gas (NO) did not induce any umuC gene expression.

Construction of frameshift mutation hot spots within the tetracycline resistance gene of pBR322

The chemical carcinogen, N-2-acetylaminofluorene (AAF) when bound covalently to DNA induces a majority (greater than 90%) of frameshift mutations. The mutations occur with high frequencies at defined sequences (i.e. mutation hot spots). Two classes of mutation hot spots were found: at repetitive sequences and at specific non-repetitive sequences. Mutations at the repetitive sequences depend upon a functional umuC gene whereas mutations at specific non-repetitive sequences are umuC-independent. The first discovered sequence of this class is the NarI restriction enzyme recognition sequence (5'GGCGCC3'). In an attempt to define a family of such sequences we constructed a related sequence 5'GCGCGC3' within the tetracycline resistance gene of pBR322. This sequence was also found to be an--AAF induced--2 frameshift mutation hot spot in both wild type and umuC strains.

Effect of adenine, cytidine and guanosine on the expression of the SOS system in Escherichia coli.

Addition of cytidine or guanosine to UV-irradiated cells of a RecA+ strain of Escherichia coli did not produce any effect on the induction of two SOS functions: inhibition of cell division and expression of the umuC gene. Under the same conditions adenine gave a slight increase in the induction of these two responses. In a RecA441 mutant growing at 42 degrees C, both cytidine and guanosine inhibited these SOS functions, whereas adenine produced a large increase in their expression. Moreover, the ATP concentration of the RecA441 mutant at 42 degrees C showed a decrease which occurred earlier in the cells growing in the presence of cytidine or guanosine than in the absence of either compound. Adenine induced an increase of about three times the initial ATP concentration of this mutant at 42 degrees C which dropped quickly after 10 min. Neither cytidine nor guanosine increased the evolution of cellular ATP in UV-irradiated cells of the RecA+ strain, whereas adenine had only a slight positive effect. However, in UV-irradiated RecA+ cells with and without adenine, ATP levels dropped quickly to the initial value after 20 min. These data suggest that the influence of adenine, cytidine and guanosine on the expression of the RecA441 phenotype at 42 degrees C may be due to alteration of the cellular ATP concentration of this mutant.

Further characterization of repair of 8-methoxypsoralen crosslinks in UV-excision-defective Escherichia coli

Evidence was previously presented for a new pathway for the repair of 8-methoxypsoralen DNA crosslinks. The pathway, which is independent of the uvrA gene but deficient in rep mutants, has now been further characterized and shown to be more active in minimal than in nutrient growth media and to be inhibited by acriflavine. Although crosslink repair is much reduced in recA bacteria, some still occurs as judged by the effect of acriflavine. By the same criterion, crosslink repair occurs in bacteria with point mutations in the uvrA and uvrB genes, in bacteria with a deletion covering the uvrB gene, and in polA uvrA bacteria. Bacteria with insertions rather than point mutations in the uvrA gene, although showing evidence of repair, demonstrated minimal inhibition with acriflavine suggesting the possibility that the uvrA gene product, even in enzymically inactive, might be able to interact with DNA lesions in the presence of acriflavine and prevent crosslink repair. Crosslink repair in E. coli WP2 uvrA is associated with base-repair substitution mutagenesis and may be characterized as an error-prone process. Crosslink in uvrA bacteria is reduced but not eliminated by a mutation in the umuC gene.