Different efficiency of UmuDC and MucAB proteins in UV light induced mutagenesis in Escherichia coli

Summary Two multicopy plasmids carrying either the umuDC or the mucAB operon were used to compare the efficiency of UmuDC and MucAB proteins in UV mutagenesis of Escherichia coli K12. It was found that in recA ⁺ uvr ⁺bacteria, plasmid pIC80, mucAB ⁺mediated UV mutagenesis more efficiently than did plasmid pSE117, umuDC ⁺. A similar result was obtained in lexA51(Def) cells, excluding the possibility that this was due to a differential regulation by LexA of the umuDC and mucAB operons. We conclude that some structural characteristic of the UmuDC and MucAB proteins determines their different efficiency in UV mutagenesis. This characteristic could be also responsible for the observation that in the recA430 mutant, pIC80 but no pSE117 can mediate UV mutagenesis. In the recA142 mutant, pIC80 also promoted UV mutagenesis more efficiently than pSE117. In this mutant, the recombination proficiency, the protease activity toward LexA and the mutation frequency were increased by the presence of adenine in the medium. In recA ⁺ uvrB5 bacteria, plasmid pSE117,umuDC caused both an increase in UV sensitivity as well as a reduction in the mutation frequency. These nagative effects resulting from the overproduction of UmuDC proteins were higher in recA142 uvrB5 than in recA ⁺ uvrB5 cells. In contrast, overproduction of MucAB proteins in excision-deficient bacteria containing pIC80 led to a large increase in the mutation frequency. We suggest that the functional differences between UmuDC and MucAB proteins might be due to their different dependence on the direct role of RecA protease in UV mutagenesis.     

Interactions between epsilon, the proofreading subunit of DNA polymerase III, and proteins involved in the SOS response of Escherichia coli

Summary Epsilon, a fidelity subunit of Escherichia coli DNA Polymerase III, is encoded by dnaQ ⁺. dnaQ49 is a recessive allele that confers temperature-sensitive and saltsuppressible phenotypes for both replication fidelity and viability. SOS mutagenesis in E. coli is regulated by LexA and requires activated RecA (RecA^*) and the products of the umuDC operon. dnaQ49 strains with various recA, lexA and umuDC alleles were constructed to determine if activities induced as part of the SOS response influence epsilon activity. We found: (1) both UmuDC and RecA^* independently enhance the dnaQ49 mutator phenotype, and (2) expression of RecA^* activity in the absence of UmuDC function increases the temperature sensitivity for viability of dnaQ49. These results support the hypothesis that RecA and one or both of the UmuDC proteins interact with the replication complex during SOS mutagenesis.     

Proteolytic processing of MucA protein in SOS mutagenesis: Both processed and unprocessed MucA may be active in the mutagenesis

Summary The mucAB operon carried on plasmid pKM101, which is an analogue of the umuDC operon of Escherichia coli, is involved in UV mutagenesis and mutagenesis induced by many chemicals. Mutagenesis dependent on either the umuDC or mucAB operon requires the function of the recA gene and is called SOS mutagenesis. By treating the cell with agents that damage DNA, RecA protein is activated by conversion into a form (RecA^*) that mediates proteolytic cleavage of the LexA repressor and derepresses the SOS genes including mucAB. Since UmuD protein is proteolytically processed to an active form (UmuD^*) in a RecA^*-dependent fashion, and MucA shares extensive amino acid homology with UmuD, we examined whether MucA is similarly processed in the cell, using antiserum against a LacZ-MucA fusion protein. Like UmuD, MucA protein is indeed proteolytically processed in a RecA^*-dependent fashion. In recA430 strains, MucAB but not UmuDC can mediate UV mutagenesis. However, MucA was not processed in the recA430 cells treated with mitomycin C. We constructed, by site-directed mutagenesis, several mutant mucA genes that encode MucA proteins with alterations in the amino acids flanking the putative cleavage site (Ala25-Gly26). MucA(Cys25) was processed and was as mutagenically active as wild-type MucA; MucA(Asp26) and MucA(Cys25,Asp26) were not processed, and were mutagenically inactive; MucA-(Thr25) was not processed, but was mutagenically as active as wild-type MucA. The mutant mucA gene that encoded the putative cleavage product of MucA was as active as mucA ⁺ in UV mutagenesis. These results raise the possibility that both the nascent MucA and the processed product are active in mutagenesis.     

Induction of only one SOS operon, umuDC, is required for SOS mutagenesis in Escherichia coli

The actions of UmuDC and RecA proteins, respectively in SOS mutagenesis are studied here with the following experimental strategy. We used lexAl (Ind^–) bacteria to maintain all SOS proteins at their basal concentrations and then selectively increased the concentration of either UmuDC or RecA protein. For this purpose, we isolated operator-constitutive mutations o ^c in the umuDC and umuD'C operons and also used the o ₉₈ ^c -recA mutation. The o ₁ ^c -umuDC mutation prevents LexA repressor from binding to the operator and improves the Pribnow box consensus sequence. As a result, 5000 UmuD and 500 UmuC molecules per cell were produced in lexAl bacteria. This concentration is sufficient to restore SOS mutagenesis. The level of RecA protein present in the repressed state promoted full UmuD cleavage. Overproduction of RecA alone did not promote SOS mutagenesis. Increasing the level of RecA in the presence of high concentrations of UmuDC proteins has no further effect on SOS mutgenesis. We conclude that, after DNA damage, umuDC is the only SOS operon that must be induced in Escherichia coli to promote SOS mutagenesis.     

Involvement of umuDCST genes in nitropyrene-induced -CG frameshift mutagenesis at the repetitive CG sequence in the hisD3052 allele of Salmonella typhimurium

Expression of the umuDC operon is required for UV and most chemical mutagenesis in Escherichia coli. The closely related species Salmonella typhimurium has two sets of umuDC-like operons, umuDC _ST on the chromosome and samAB on a 60-MDa cryptic plasmid. The roles of theumuDC-like operons in chemically induced frameshift mutagenesis of the hisD3052 allele of S. typhimurium were investigated. Introduction of a pBR322-derived plasmid carrying umuDCST increased the rate of reversion of hisD3052, following treatment with 1-nitropyrene (1-NP) or 1,8-dinitropyrene (1,-8DNP) tenfold and fivefold, respectively, whereas it did not substantially increase the rate of reversion induced by other frameshift mutagens, i.e. 2-nitrofluorene (2NF) and 2-amino- 3-methyldipyrido[1,2-a:3 ,2-d]imi-dazole (Glu-P-1). Introduction of a pBR322-derived plasmid carrying samAB did not increase the incidence of reversion of hisD3052 observed with any of the mutagens examined. Deletion of umuDC _STSubstantially lowered the reversion rate induced by l-NP or 1,8-DNP, but it did not affect reversion induced by 2-NF, Glu-P-1 or N-hydroxyacetylaminofluorene (N-OH-AAF). Deletion of samAB had little impact on reversion incidence induced by any of the five frameshift mutagens. DNA amplification using the polymerase chain reaction technique followed by restriction enzyme analysis using BssHII, suggested that the mutations induced by the five frameshift mutagens were all CG deletions at the CGCGCGCG sequence in hisD3052. These results suggest that umuDCST, but not samAB, is involved in the -2 frameshift mutagenesis induced by l-NP and 1,8-DNP at the repetitive CG sequence, whereas neither operon participates in induction of the same type of mutations by 2-NF, Glu-P-1 or N-OH-AAF.     

The spectra of base substitutions induced by the impCAB, mucAB and umuDC error-prone DNA repair operons differ following exposure to methyl methanesulfonate

We have used the lacZ reversion assay to study the mutation spectra induced by the Escherichia coli chromosomal umuDC operon and of its two plasmid-borne analogues impCAB and mucAB following exposure of cells to UV light and methyl methane-sulfonate (MMS). We have shown that the impCAB, mucAB and umuDC operons all produce a similar response to UV light which results almost exclusively in AT GC transitions. However, we found that the three operons produced different responses to alkylating agents. We found that with MMS the chromosomal umuDC operon produced almost exclusively AT GC transitions, whilst both mucAB and impCAB produced predominantly transversions. In the case of the impCAB operon the mutation spectrum contained more AT TA than GC TA transversions; this balance was reversed with mucAB. The effect of the copy number of the error-prone DNA repair operons upon the mutagenic spectra was also studied. The results obtained suggest that the copy number of the imp operon does not greatly affect the specificity of base substitutions observed. However, an increase in the copy number of the umuDC operon greatly affected the specificity of base substitution, such that virtually no transitions were produced and the spectrum was dominated by GC/AT TA transversions. It appears that the three error-prone DNA repair operons impCAB, mucAB and umuDC, despite showing strong structural and functional homologies, can display major differences in the spectrum of base changes induced during mutagenesis. We propose that the type of misincorporation/chain extension which DNA polymerase III is allowed to synthesize on a damaged DNA template is extremely sensitive to both the amount and type of error-prone repair proteins present. The modulation of these events by the different proteins can result in widely different mutagenic changes in the repaired DNA.     

Induction of only one SOS operon,umuDC, is required for SOS mutagenesis in Escherichia coli

The actions of UmuDC and RecA proteins, respectively in SOS mutagenesis are studied here with the following experimental strategy. We used lexAl (Ind^?) bacteria to maintain all SOS proteins at their basal concentrations and then selectively increased the concentration of either UmuDC or RecA protein. For this purpose, we isolated operator-constitutive mutations o ^c in the umuDC and umuD'C operons and also used the o ₉₈ ^c -recA mutation. The o ₁ ^c -umuDC mutation prevents LexA repressor from binding to the operator and improves the Pribnow box consensus sequence. As a result, 5000 UmuD and 500 UmuC molecules per cell were produced in lexAl bacteria. This concentration is sufficient to restore SOS mutagenesis. The level of RecA protein present in the repressed state promoted full UmuD cleavage. Overproduction of RecA alone did not promote SOS mutagenesis. Increasing the level of RecA in the presence of high concentrations of UmuDC proteins has no further effect on SOS mutgenesis. We conclude that, after DNA damage, umuDC is the only SOS operon that must be induced in Escherichia coli to promote SOS mutagenesis.     

Different UmuC requirements for generation of different kinds of UV-induced mutations in Escherichia coli

An Escherichia coli strain bearing the dnaQ49 mutation, which results in a defective s subunit of DNA polymerase III, and carrying the lexA71 mutation, which causes derepression of the SOS regulon, is totally unable to maintain high-copy-number plasmids containing the umuDC operon. The strain is also unable to maintain the pAN4 plasmid containing a partial deletion of the umuD gene but retaining the wild-type umuC gene. These results suggest that a high cellular level of UmuC is exceptionally harmful to the defective DNA polymerase III of the dnaQ49 mutant. We have used this finding as a basis for selection of new plasmid umuC mutants. The properties of two such mutants, bearing the umuC61 or umuC95 mutation, are described in detail. In the umuC122:: Tn 5 strain harbouring the mutant plasmids, UV-induced mutagenesis is severely decreased compared to that observed with the parental umuDC ⁺ plasmid. Interestingly, while the frequency of UV-induced GC AT transitions is greatly reduced, the frequency of AT TA transversions is not affected. Both mutant plasmids bear frameshift mutations within the same run of seven A residues present in umuC ⁺; in umuC61 the run is shortened to six A whereas in umuC95 is lengthened to eight A. We have found in both umuC61 and umuC95 that translation is partially restored to the proper reading frame. We propose that under conditions of limiting amounts of UmuC, the protein preferentially facilitates processing of only some kinds of UV-induced lesions.     

Role of the RecF gene product in UV mutagenesis of lambda phage

Summary E. coli recF mutants have a greatly reduced capacity for Weigle mutagenesis of ultraviolet light-irradiated lambda phage. A recF 332::Tn3 mutation was introduced into an E. coli recA441 lexA51 strain which constitutively expresses SOS functions. Weigle mutagenesis of phage lambda could occur in the resulting strain in the absence of host cell irradiation, and was increased when the recA441 (tif) allele was activated by increased temperature and excess adenine. The inability of recF strains to support Weigle mutagenesis can therefore be ascribed to a defect in expression of SOS functions after irradiation.     

Analysis of the mutagenic properties of the UmuDC, MucAB and RumAB proteins, using a site-specific abasic lesion

ThemucAB andrumAB loci have been shown to promote mutagenesis to a greater extent than the structurally and functionally homologousEscherichia coli umuDC operon. We have analyzed the basis of this enhanced mutagenesis by comparing the influence of these operons, relative toumuDC, on the mutagenic properties of each of two abasic sites, specifically located in a single-stranded vector. Experiments with these vectors are useful analytical tools because they provide independent estimates of the efficiency of translesion synthesis and of the relative frequencies of each type of nucleotide insertion or other kind of mutagenic event. TheumuDC, mucAB, andrumAB genes were expressed from their naturalLexA-regulated promoter on low-copy-number plasmids in isogenic strains carrying aumuDC deletion. In addition, plasmids expressing the UmuD'C, MucA'B, or RumA'B proteins were also used. Compared toumuDC, the chief effect ofmucAB was to increase the efficiency of translesion synthesis past the abasic site. The enhanced capacity ofmucAB for translesion synthesis depended about equally on an inherently greater capacity to promote this process and on a greater susceptibility of the MucA protein to proteolytic processing. The RumA protein also appeared to be more susceptible to proteolytic processing, but the inherent capacity of theRum products for translesion synthesis was no greater than that ofUmuDC. dAMP was inserted opposite one of the two abasic sites studied at a somewhat greater frequency in strains expressingrum (82%) compared to those expressingumu (72%), which might result in higher mutation frequencies inrumAB than inumuDC strains.     

Differential repression of SOS genes by unstable LexA41 (Tsl-1) protein causes a “split-phenotype” in Escherichia coli K-12

The lexA41 (formerly tsl-1) mutant was isolated as an ultraviolet light-resistant, temperature-sensitive derivative of its ultraviolet light-sensitive lexA3(Ind⁻) parent. Cells exhibit a so-called “split-phenotype”, a phenomenon in which only a subset of the SOS responses can be detected physiologically following inducing treatments. lexA41 has been cloned and sequenced; the mutant gene retains the (tflexA3) mutation (Gly to Asp at position 85) and has a second mutation, lexA41 (Ala to Thr at position 131). We show that LexA41 protein is not cleaved by the RecA protein-catalyzed pathway in vivo, but the mutant protein is degraded by the Lon protease at both 32 ° C and 42 ° C. β-Galactosidase activities of lac fusions to 13 different SOS promoters were measured at 30 ° C and 42 ° C to determine levels of expression and were found to vary considerably. The temperature-sensitive phenotype is a result of increased expression of sulA, which encodes a division inhibitor, at 42 ° C. Excision repair genes, including uvn A, uvrB and uvr D, are constitutively expressed at 30 ° C accounting for the ultraviolet light resistance of the lexA41 mutant, but the SOS mutagenesis operon, umuD,C, is not adequately derepressed, thereby explaining the failure to induce mutagenesis in this background. This differential expression of SOS genes gives a plausible explanation of the split-phenotype associated with lexA41.     

New mutations in cloned Escherichia coli umuDC genes: Novel phenotypes of strains carrying a umuC125 plasmid

The umuDC locus of Escherichia coli is required for most mutagenesis by UV and many chemicals. Mutations in E. coli umuDC genes cloned on pBR322-derived plasmids wer e isolated by two methods. First, spontaneously-arising mutant umuDC plasmids that failed to confe cold-sensitive growth on a lexA51(Def) strain were isolated by selection. Second, mutant umuDC plasmids that affected apparent mutant yield after UV-irradiation in a strain carrying umuD⁺C⁺ in the chromosome were isolated by screening hydroxylamine-mutagenized umuD⁺C⁺ plasmids. pBR322-derived umuD⁺C⁺ plasmids inhibited the induction of the SOS response of lexA⁺ strains as measured by expression of din::Mu dl(lac) Ap) fusionsbut most mutant plasmids did not. Mutant plasmids defective in complementation of chromosomal umuD44, umuC36, or both were found among those selected for failure to confer cold-sensitivity, whereas those identified by the screening procedure yielded mostly mutant plasmids with more complex phenotypes. We studied in greater detail a plasmid pLM109, carrying the umuC125 mutation. This plasmid increased the sensitivity of lexA⁺ strainsto killing by UV-irradiation but was able to complement the deficiencies of umuC mutants in UV mutagenesis. pLM109 failed to confer cold-sensitive growth on lexA(Def) strains but inhibited SOS induction in lexA⁺ strains. The effect of pLM109 on the UV sensitivity of lexA(Def)strains was similar to that of the parental umuD⁺C⁺ plasmid. The mutation responsible for the phenotypes of pLM109 was localized to a 615-bp fragment. DNA sequencing revealed that the umuC125mutation was a G:C → A:T transition that changed codon 39 of umuC from GCC → GTC thus changing Ala39 to Val39. The implications of the umuC125 mutation for umuDC-dependent effects on UV-mutagenesis and cell survival after UV damage are discussed.     

How Escherichia coli sets different basal levels in SOS operons

The recA and sfiA genes of Escherichia coli are SOS operons regulated negatively by the LexA repressor. The steady state level of expression of recA is 10-fold higher than that of sfiA, as measured by means of recA::lac and sfiA::lac operon fusions. To study the molecular basis of this difference, we have compared the expression of these two operons in strains in which the concentration of LexA repressor was normal (lexA+), zero (spr amber mutation) or higher than normal (plasmid pJL45, carrying the lexA gene linked to the lac promoter). The results indicate (i) that the recA promoter is about 4 times stronger than the sfiA promoter (as measured in the spr strains), (ii) that neither operon has a physiologically significant level of lexA-independent expression (pJL45 strains), and (iii) that the recA operator has about 2.5 times lower affinity than the sfiA operator for LexA repressor (comparison of lex+ and spr strains). Considering our previous results that the sfiA operon (high operator affinity of LexA) is derepressed very rapidly after inducing treatments and that the recA operon (low operator affinity) is repressed very rapidly when induction is stopped, we conclude that differences in operator affinity do not affect inducibility but serve only to set the basal levels of the different SOS functions.     

Functional complementation between chromosomal and plasmid mutagenic DNA repair genes in bacteria

Summary The umuDC operons of Escherichia coli and Salmonella typhimurium and the analogous plasmid operons mucAB and impCAB have been previously characterized in terms of their roles in DNA repair and induced mutagenesis by radiation and many chemicals. The interrelationships of these mutagenic DNA repair operons were examined in vivo in functional tests of interchangeability of operon subunits in conferring UV resistance and UV mutability phenotypes to wild-type S. typhimurium and umu mutants of E. coli. This approach was combined with DNA and protein sequence comparisons between the four operons and a fifth operon, samAB, from the S. typhimurium LT2 cryptic plasmid. Components of the E. coli and S. typhimurium umu operons were reciprocally interchangeable whereas impCA and mucA could not function with umuC in either of these species. mucA and impB could also combine to give a mutagenic response to UV. These active combinations were associated with higher degrees of conservation of protein sequence than in other heterologous gene combinations and related to specific regions of sequence that may specify subunit interactions. The dominance of the E. coli umuD44 mutation over umuD was revealed in both wild-type E. coli and S. typhimurium and also demonstrated against impCAB. Finally interspecies transfer showed that the apparently poor activity of the S. typhimurium umuD gene in situ is not the result of an inherent defect in umuD but is due to the simultaneous presence of the S. typhimurium umuC sequence. It is suggested that the limitation of umuD activity by umuC in S. typhimurium is the basis of the poor induced mutability of this organism.     

Identification of mucAB-like homologs on two IncT plasmids, R394 and Rts-1

Recent phylogenetic analysis of the superfamily of lesion-replicating DNA polymerases suggest that they can be broadly divided into four sub-groups comprised of UmuC-like, DinB-like, Rev1-like and Rad30-like proteins. The UmuC-like sub-family is best characterized at the genetic level and sequence analysis of eleven umu orthologs, residing on bacterial chromosomes or on self-transmissible R-plasmids allows further subdivision into five sub-groups (UmuDC, MucAB, ImpAB, RumAB and RulAB) based on amino acid sequence conservation. Some of these orthologs are apparently inactive in situ, but may promote increased mutagenesis and survival when subcloned and expressed from high-copy number plasmids. We were, therefore, interested in devising an assay that would identify umuC-like genes in situ in the absence of a functional assay. To this end, degenerate primers directed towards conserved amino acid regions within the UmuC-like sub-family of DNA polymerases were designed and used to identify mucAB-like operons on the IncT plasmids, R394 and Rts-1.Interestingly, DNA sequence analysis of an 7 kb region of R394 identified two LexA-regulated genes immediately downstream of mucAB_(R394) that are similar to the chromosomally-encoded Escherichia coli tus gene and the IncI plasmid-encoded impC gene, respectively. Analysis of the R394 and Rts-1 mucB genes revealed that both contain insertions which result in the expression of a truncated inactive MucB protein. While R394 was unable to restore mutagenesis functions to a ΔumuDC E. coli strain, Rts-1 surprisingly promoted significant levels of MMS-induced SOS mutagenesis, raising the possibility that Rts-1 encodes another, yet unidentified, umu-like homolog.     

The spectra of base substitutions induced by the impCAB,mucAB and umuDC error-prone DNA repair operons differ following exposure to methyl methanesulfonate

We have used the lacZ reversion assay to study the mutation spectra induced by the Escherichia coli chromosomal umuDC operon and of its two plasmid-borne analogues impCAB and mucAB following exposure of cells to UV light and methyl methane-sulfonate (MMS). We have shown that the impCAB, mucAB and umuDC operons all produce a similar response to UV light which results almost exclusively in AT → GC transitions. However, we found that the three operons produced different responses to alkylating agents. We found that with MMS the chromosomal umuDC operon produced almost exclusively AT → GC transitions, whilst both mucAB and impCAB produced predominantly transversions. In the case of the impCAB operon the mutation spectrum contained more AT → TA than GC → TA transversions; this balance was reversed with mucAB. The effect of the copy number of the error-prone DNA repair operons upon the mutagenic spectra was also studied. The results obtained suggest that the copy number of the imp operon does not greatly affect the specificity of base substitutions observed. However, an increase in the copy number of the umuDC operon greatly affected the specificity of base substitution, such that virtually no transitions were produced and the spectrum was dominated by GC/AT → TA transversions. It appears that the three error-prone DNA repair operons impCAB, mucAB and umuDC, despite showing strong structural and functional homologies, can display major differences in the spectrum of base changes induced during mutagenesis. We propose that the type of misincorporation/chain extension which DNA polymerase III is allowed to synthesize on a damaged DNA template is extremely sensitive to both the amount and type of error-prone repair proteins present. The modulation of these events by the different proteins can result in widely different mutagenic changes in the repaired DNA.     

Viability of Escherichia coli K-12 DNA adenine methylase (dam) mutants requires increased expression of specific genes in the SOS regulon

Summary We have examined the level of expression of the SOS regulon in cells lacking DNA adenine methylase activity (dam ^-). Mud (Ap, lac) fusions to several SOS operons (recA, lexA, uvrA, uvrB, uvrD, sulA, dinD and dinF) were found to express higher levels of -galactosidase in dam ^- strains than in isogenic dam ⁺ strains. The attempted construction of dam ^- strains that were also mutant in one of several SOS genes indicated that the viability of methylase-deficient strains correlates with the inactivation of the SOS repressor (LexA protein). Consistent with this, the wild-type functions of two LexA-repressed genes (recA and ruv) appear to be required for dam ^- strain viability.     

Constitutive expression of the SOS response in recA718 mutants of Escherichia coli requires amplification of RecA718 protein.

In recA718 lexA+ strains of Escherichia coli, induction of the SOS response requires DNA damage. This implies that RecA718 protein, like RecA+ protein, must be converted, by a process initiated by the damage, to an activated form (RecA) to promote cleavage of LexA, the cellular repressor of SOS genes. However, when LexA repressor activity was abolished by a lexA-defective mutation [lexA(Def)], strains carrying the recA718 gene (but not recA+) showed strong SOS mutator activity and were able to undergo stable DNA replication in the absence of DNA damage (two SOS functions known to require RecA activity even when cleavage of LexA is not necessary). lambda lysogens of recA718 lexA(Def) strains exhibited mass induction of prophage, indicative of constitutive ability to cleave lambda repressor. When the cloned recA718 allele was present in a lexA+ strain on a plasmid, SOS mutator activity and beta-galactosidase synthesis under LexA control were expressed in proportion to the plasmid copy number. We conclude that RecA718 is capable of becoming activated without DNA damage for cleavage of LexA and lambda repressor, but only if it is amplified above its base-line level in lexA+ strains. At amplified levels, RecA718 was also constitutively activated for its roles in SOS mutagenesis and stable DNA replication. The nucleotide sequence of recA718 reveals two base substitutions relative to the recA+ sequence. We propose that the first allows the protein to become activated constitutively, whereas the second partially suppresses this capability.     

Alleviation of EcoK DNA restriction in Escherichia coli and involvement of umuDC activity.

Summary The activity of the EcoK DNA restriction system of Escherichia coli reduces both the plating efficiency of unmodified phage and the transforming ability of unmodified pBR322 plasmid DNA. However, restriction can be alleviated in wild-type cells, by UV irradiation and expression of the SOS response, so that 10³-to 10⁴-fold increases in phage growth and fourfold increases in plasmid transformation occurred with unmodified DNA. Restriction alleviation was found to be a transient effect because induced cells, which initially failed to restrict unmodified plasmid DNA, later restricted unmodified phage . Although the SOS response was needed for restriction alleviation, constitutive SOS induction, elicited genetically with a recA730 mutation, did not alleviate restriction and UV irradiation was still needed. A hitherto unsuspected involvement of the umuDC operon in this alleviation of restriction is characterized and, by differential complementation, was separated from the better known role of umuDC in mutagenic DNA repair. The need for cleavage of UmuD for restriction alleviation was shown with plasmids encoding cleavable, cleaved, and non-cleavable forms of UmuD. However, UV irradiation was still needed even when cleaved UmuD was provided. The possibility that restriction alleviation occurs by a general inhibition of the EcoK restriction/modification complex was tested and discounted because modification of was not reduced by UV irradiation. An alternative idea, that restriction activity was competitively reduced by an increase in EcoK modification, was also discounted by the lack of any increase in the modification of Ral^–, a naturally undermodified phage. Other possible mechanisms for restriction alleviation are discussed.     

Involvement of umuDC ST genes in nitropyrene-induced -CG frameshift mutagenesis at the repetitive CG sequence in the hisD3052 allele of Salmonella typhimurium

Expression of the umuDC operon is required for UV and most chemical mutagenesis in Escherichia coli. The closely related species Salmonella typhimurium has two sets of umuDC-like operons, umuDC _ST on the chromosome and samAB on a 60-MDa cryptic plasmid. The roles of theumuDC-like operons in chemically induced frameshift mutagenesis of the hisD3052 allele of S. typhimurium were investigated. Introduction of a pBR322-derived plasmid carrying umuDCST increased the rate of reversion of hisD3052, following treatment with 1-nitropyrene (1-NP) or 1,8-dinitropyrene (1,-8DNP) tenfold and fivefold, respectively, whereas it did not substantially increase the rate of reversion induced by other frameshift mutagens, i.e. 2-nitrofluorene (2NF) and 2-amino- 3-methyldipyrido[1,2-a:3 ′,2′-d]imi-dazole (Glu-P-1). Introduction of a pBR322-derived plasmid carrying samAB did not increase the incidence of reversion of hisD3052 observed with any of the mutagens examined. Deletion of umuDC _STSubstantially lowered the reversion rate induced by l-NP or 1,8-DNP, but it did not affect reversion induced by 2-NF, Glu-P-1 or N-hydroxyacetylaminofluorene (N-OH-AAF). Deletion of samAB had little impact on reversion incidence induced by any of the five frameshift mutagens. DNA amplification using the polymerase chain reaction technique followed by restriction enzyme analysis using BssHII, suggested that the mutations induced by the five frameshift mutagens were all CG deletions at the CGCGCGCG sequence in hisD3052. These results suggest that umuDCST, but not samAB, is involved in the -2 frameshift mutagenesis induced by l-NP and 1,8-DNP at the repetitive CG sequence, whereas neither operon participates in induction of the same type of mutations by 2-NF, Glu-P-1 or N-OH-AAF.