Role of the E. coli umuC gene product in the repair of single-stranded DNA phage

The umuC product of Escherichia coli has been suggested to have a central role in SOS induced error prone replication of DNA (Kato and Shinoura 1977). To investigate this possibility, we examined the effect of umuC mutations on error prone repair of single and double-stranded DNA phages. No Weigle reactivation of M13 phage was detected in a umuC mutant. Reactivation of lambda phage was reduced but still evident. However mutagenesis occurred in both cases. These results suggest that induced error prone replication of phage DNA can occur via umuC dependent (transdimer synthesis) and umuC independent mechanisms.     

Mechanisms of frameshift mutagenesis by aflatoxin B1-2,3-dichloride

In order to characterize frameshift mutagenesis by aflatoxin B1-2,3-dichloride (AFB1Cl2), we have introduced a +1 (BK8) or a -1 (HS8) frameshift within the lacZ alpha gene segment contained in the phage M13mp8 to obtain lacZ alpha- derivatives. BK8 or HS8 replicative form DNA was modified with AFB1Cl2 in vitro, transfected into appropriate Escherichia coli hosts and lacZ alpha+ revertants scored and defined by DNA sequencing. The -1 frameshift (BK8) results suggest the following. (1) The E. coli recA gene is not absolutely required for AFB1Cl2-induced frameshift mutagenesis; however, in recA+ cells, ultraviolet light (SOS) induction enhances AFB1Cl2 mutagenesis, but such ultraviolet induction is not required. The plasmid pGW270 (mucAB+) significantly enhances the AFB1Cl2-induced frameshift mutagenesis. The uvrABC+ excision system plays a major role in the repair of AFB1Cl2-induced damage. (2) Sequence analysis reveals that AFB1Cl2 induces two classes of -1 frameshift mutations: the simple class in which the frameshift is due to the loss of one base-pair, and the complex class in which the loss of a base-pair is coupled to a vicinal base substitution. Both types of mutations occur predominantly at G.C runs, which are hotspots for AFB1Cl2 damage. The complex mutations appear to be concerted events targeted by a single AFB1Cl2 adduct. The frequency of these complex mutations is significantly enhanced by mucAB activity. In this system, recA activity is required for generation of significant levels of complex mutations. An analysis of the +1 frameshifts (HS8) reveals that AFB1Cl2 induces +1 frameshifts with an efficiency comparable to that for -1 frameshifts. Most +1 frameshifts occur by the addition of a base, and a third of the additions are complex mutations because they are accompanied by at least one base substitution. All simple additions occur at G.C runs; however, in a striking contrast to spontaneous insertions, a majority of the induced events introduce an A.T pair at these sites. Our data suggest a model for the generation of base substitution as well as simple and complex frameshift mutations induced by AFB1Cl2. To the extent determined, the frameshift specificity of aflatoxin B1 activated by metabolic enzymes is similar to that of AFB1Cl2.     

SOS induction in Escherichia coli by infection with mutant filamentous phage that are defective in initiation of complementary-strand DNA synthesis.

We report that the SOS response is induced in Escherichia coli by infection with mutant filamentous phage that are defective in initiation of the complementary (minus)-strand synthesis. One such mutant, R377, which lacks the entire region of the minus-strand origin, failed to synthesize any detectable amount of primer RNA for minus-strand synthesis. In addition, the rate of conversion of parental single-stranded DNA of the mutant to the double-stranded replicative form in infected cells was extremely slow. Upon infection, R377 induced the SOS response in the cell, whereas the wild-type phage did not. The SOS induction was monitored by (i) induction of beta-galactosidase in a strain carrying a dinD::lacZ fusion and (ii) increased levels of RecA protein. In addition, cells infected with R377 formed filaments. Another deletion mutant of the minus-strand origin, M13 delta E101 (M. H. Kim, J. C. Hines, and D. S. Ray, Proc. Natl. Acad. Sci. USA 78:6784-6788, 1981), also induced the SOS response in E. coli. M13Gori101 (D. S. Ray, J. C. Hines, M. H. Kim, R. Imber, and N. Nomura, Gene 18:231-238, 1982), which is a derivative of M13 delta E101 carrying the primase-dependent minus-strand origin of phage G4, did not induce the SOS response. These observations indicate that single-stranded DNA by itself induces the SOS response in vivo.     

Non-targeted mutagenesis of unirradiated lambda phage in Escherichia coli host cells irradiated with ultraviolet light

Non-targeted mutagenesis of lambda phage by ultraviolet light is the increase over background mutagenesis when non-irradiated phage are grown in irradiated Escherichia coli host cells. Such mutagenesis is caused by different processes from targeted mutagenesis, in which mutations in irradiated phage are correlated with photoproducts in the phage DNA. Non-irradiated phage grown in heavily irradiated uvr+ host cells showed non-targeted mutations, which were 3/4 frameshifts, whereas targeted mutations were 2/3 transitions. For non-targeted mutagenesis in heavily irradiated host cells, there were one to two mutant phage per mutant burst. From this and the pathways of lambda DNA synthesis, it can be argued that non-targeted mutagenesis involves a loss of fidelity in semiconservative DNA replication. A series of experiments with various mutant host cells showed a major pathway of non-targeted mutagenesis by ultraviolet light, which acts in addition to "SOS induction" (where cleavage of the LexA repressor by RecA protease leads to din gene induction): (1) the induction of mutants has the same dependence on irradiation for wild-type and for umuC host cells; (2) a strain in which the SOS pathway is constitutively induced requires irradiation to the same level as wild-type cells in order to fully activate non-targeted mutagenesis; (3) non-targeted mutagenesis occurs to some extent in irradiated recA recB cells. In cells with very low levels of PolI, the induction of non-targeted mutagenesis by ultraviolet light is enhanced. We propose that the major pathway for non-targeted mutagenesis in irradiated host cells involves binding of the enzyme DNA polymerase I to damaged genomic DNA, and that the low polymerase activity leads to frameshift mutations during semiconservative DNA replication. The data suggest that this process will play a much smaller role in ultraviolet mutagenesis of the bacterial genome than it does in the mutagenesis of lambda phage.     

60 years of SOS repair

This review integrates 60 years of research on SOS repair and SOS mutagenesis in prokaryotes and eukaryotes, from Jean Weigle’s experiment in 1953 (mutagenesis of lambda bacteriophage in UV-irradiated bacteria) to the latest achievements in studying SOS mutagenesis on all living organisms, i.e., Eukarya, Archaea and Bacteria. A key role in establishing of a biochemical basis for SOS mutagenesis belongs to the finding in 1998–1999 that specific error-prone DNA polymerases (PolV and others) catalyzed translesion synthesis on damaged DNA. This review focuses on recent studies that address new models for SOS-induced mutagenesis in Escherichia coli and Homo sapiens cells.     

Insertion of the Tn3 transposon into the genome of the single-stranded DNA phage M13.

The transposable genetic element Tn3, which carries an ampicillin (Ap) resistance determinant, has been translocated from a ColE1-Apr plasmid, RSF2124, to the genome of the filamentous single-stranded DNA phage M13. The site orientation of the inserted element has been determined for one such phage, M13::Tn3-15. The insertion is within the intergenic space separating genes 2 and 4 and containing both the viral strand and complementary strand origins. The lengths of both the filamentous phage and the duplex replicative form (RF) DNA are 1.7--1.8 times those of M13 phage and replicative form DNA. Both plaque formation and transduction of sensitive cells to ampicillin resistance by M13::Tn3-15 are sensitive to purified antibodies to the M13 major coat protein.     

SOS repair can be about as effective for single-stranded DNA as for double-stranded DNA and even more so.

As ordinarily measured, the SOS repair of damaged DNA by Weigle reactivation appears to be more effective for double-stranded (ds) than for single-stranded (ss) DNA bacteriophages. A complicating feature, which is usually not considered, is the possibility of DNA-protein cross-linking of ssDNA to the viral capsid, which would conceivably be an extraneous source of nonreactivable lesions. This idea is supported in studies of phage S13 by the observation that photoreactivation more than doubles when naked ssDNA is substituted for encapsidated ssDNA as the UV target. The same effect was observed for Weigle reactivation; there was little, if any, difference in the reactivation of ssDNA and dsDNA when naked DNA was irradiated. Moreover, in a uvrA mutant, ssDNA actually had the advantage; Weigle reactivation was then more than twice as effective for ssDNA as for dsDNA. It is also shown that when a suitable measure of Weigle mutagenesis is used, there is no convincing evidence that dsDNA is mutagenized more effectively than ssDNA.     

Translesion synthesis is the main component of SOS repair in bacteriophage lambda DNA.

Agents that interfere with DNA replication in Escherichia coli induce physiological adaptations that increase the probability of survival after DNA damage and the frequency of mutants among the survivors (the SOS response). Such agents also increase the survival rate and mutation frequency of irradiated bacteriophage after infection of treated bacteria, a phenomenon known as Weigle reactivation. In UV-irradiated single-stranded DNA phage, Weigle reactivation is thought to occur via induced, error-prone replication through template lesions (translesion synthesis [P. Caillet-Fauquet, M: Defais, and M. Radman, J. Mol. Biol. 117:95-112, 1977]). Weigle reactivation occurs with higher efficiency in double-stranded DNA phages such as lambda, and we therefore asked if another process, recombination between partially replicated daughter molecules, plays a major role in this case. To distinguish between translesion synthesis and recombinational repair, we studied the early replication of UV-irradiated bacteriophage lambda in SOS-induced and uninduced bacteria. To avoid complications arising from excision of UV lesions, we used bacterial uvrA mutants, in which such excision does not occur. Our evidence suggests that translesion synthesis is the primary component of Weigle reactivation of lambda phage in the absence of excision repair. The greater efficiency in Weigle reactivation of double-stranded DNA phage could thus be attributed to some inducible excision repair unable to occur on single-stranded DNA. In addition, after irradiation, lambda phage replication seems to switch prematurely from the theta mode to the rolling circle mode.     

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.     

Mechanisms of mutagenesis by the vinyl chloride metabolite chloroacetaldehyde. Effect of gene-targeted in vitro adduction of M13 DNA on DNA template activity in vivo and in vitro

2-Chloroacetaldehyde (CAA), a metabolite of the carcinogenic industrial chemical vinyl chloride, reacts with single-stranded DNA to form the cyclic etheno lesions predominantly at adenine and cytosine. In both ethenoadenine and ethenocytosine, normal Watson-Crick hydrogen-bonding atoms are compromised. We have recently shown that CAA adduction leads to efficient mutagenesis in Escherichia coli predominantly at cytosines, and less efficiently at adenines. About 80% of the mutations at cytosines were C-to-T transitions, and the remainder were C-to-A transversions, a result similar to that of many noninstructional DNA lesions opposite which adenine residues are preferentially incorporated. It is widely believed that noninstructional lesions stop replication and depend on SOS functions for efficient mutagenesis. We have examined the effects of in vitro CAA adduction of the lacZ alpha gene of phage M13AB28 on in vivo mutagenesis in SOS-(UV)-induced E. coli. CAA adduction was specifically directed to a part of the lacZ sequence within M13 replicative form DNA by a simple experimental strategy, and the DNA was transfected into appropriate unirradiated or UV-irradiated cells. Mutant progeny were defined by DNA sequencing. In parallel in vitro experiments, the effects of CAA adduction on DNA replication by E. coli DNA polymerase I large (Klenow) fragment were examined. Our data do not suggest a strong SOS dependence for mutagenesis at cytosine lesions. While adenine lesions remain much less mutagenic than cytosine lesions, mutation frequency at adenines is increased by SOS. SOS induction does not significantly alter the specificity of base changes at cytosines or adenines.(ABSTRACT TRUNCATED AT 250 WORDS)     

Site-specific mutagenesis in vivo by single methylated or deaminated purine bases

The technique of site-directed mutagenesis has been used to investigate the mutagenicity of O6-methylguanine (O6-MeG) or hypoxanthine introduced as a single lesion at a specific locus in an M13mp9 RF molecule constructed in vitro. Following transformation of O6-MeG-containing RF molecules into E. coli JM101, mutant progeny phage were produced at a frequency not significantly different from that observed with wild-type M13mp9 RF. The mutant yield was greatly enhanced by exhausting cellular O6-MeG DNA-methyltransferase before transformation. In contrast, hypoxanthine exhibited miscoding mutagenesis in the absence of interference with cellular repair mechanisms. This indicates that cellular hypoxanthine-DNA glycosylase acts inefficiently in the removal of hypoxanthine from DNA in vivo. The precise mutational changes induced by hypoxanthine were determined by DNA sequence analysis.     

Mutagenesis of ultraviolet-irradiated lambda phage by host cell irradiation: induction of Weigle mutagenesis is not an all-or-none process

Summary Ultraviolet mutagenesis of lambda phage to clear plaque formers is the same in the total phage population and in subpopulations of phage which have also mutated to gam ^- or at an amber codon. This is true for phage assayed in host cells in which Weigle mutagenesis has been either partially induced by low levels of ultraviolet irradiation, or fully induced by higher levels. If induction of Weigle mutagenesis were all-or-none, clear plaque formers in phage subpopulations selected for another mutation elsewhere would come mainly from induced cells; then the clear plaque mutation rate would always be that for fully induced host cells. Therefore, induction requires more than one lesion in host cell DNA.Although thymine starvation of cells induces synthesis of recA protein, it does not induce Weigle mutagenesis; in fact starvation inhibits induction of this process on subsequent ultraviolet irradiation of the cells.     

Genetic control of the UV-induced SOS mutator effect in single- and double-stranded DNA phages

The SOS hypothesis postulated that the mutator effect on undameged DNA that generates phage-untargeted mutagenesis (UTM) results directly from the mechanism of targeted mutagenesis. RecA protein, which stimulates the cleavage of both the LexA repressor and UmuD protein, and the UmuDC gene products are required for UV-induced targeted mutagenesis. The use of phage λ for analyzing UV-induced mutagenesis has permitted a distinction to be made between the mechanisms of targeted and untargeted mutagenesis, in that the two processes differ with respect to their genetic requirements for recA⁺ and umuDC⁺ genes. In this paper, we show thet (i) proficiency for excision repair is required for UTM in double-stranded DNA phage but not in single-stranded DNA phage; (ii) the umuC function, which is not required for UTM of the double-stranded DNA phage λ, is necessary for untargeted mutagenesis of the single-stranded DNA phages M13 and φX174; (iii) for both single-stranded and double-stranded DNA phage, UV irradiation of the host increases the level of recA730-induced UTM. Our results are also consistent with the interpretation that the expression of untargeted mutagenesis in phage λ and in M13 depends on the polymerase and to a lesser extent on the exonuclease 5′ → 3′, activities of Po1I. These results suggest that the involvement of the RecA and UmuDC proteins may be related to more than the presence of base damage in the DNA substrate.     

Survival and mutagenesis of bacteriophage T7 damaged by methyl methanesulfonate and ethyl methanesulfonate

We have examined survival and mutagenesis of bacteriophage T7 after exposure to the alkylating agents methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS). It was found that although both alkylating agents caused increased reversion of specific T7 mutations, EMS caused a higher frequency of reversion than did MMS. Exposure of the host cells to ultraviolet light so as to induce the SOS system resulted in increased survival (Weigle reactivation) of T7 phage damaged with either EMS or MMS. However, after SOS induction of the host we did not detect an accompanying increase in mutation frequency measured as either reversion of specific T7 mutants or by generation of mutations in the T7 gene that codes for phage ligase. Neither mutation frequency nor survival of alkylated phage was affected by the umuD,C mutation in the Escherichia coli host nor by the presence of plasmid pKM101. This may mean that the mode of Weigle reactivation that is detected in T7 is not mutagenic in nature.     

Escherichia coli cells bearing mutA, a mutant glyV tRNA gene, express a recA-dependent error-prone DNA replication activity

A base substitution mutation (mutA) in the Escherichia coli glyV tRNA gene potentiates asp --> gly mistranslation and confers a strong mutator phenotype that is SOS independent, but requires recA, recB and recC genes. Here, we demonstrate that mutA cells express an error-prone DNA polymerase by using an in vitro experimental system based on the conversion of phage M13 single-stranded viral DNA bearing a model mutagenic lesion to the double-stranded replicative form. Amplification of the newly synthesized strand followed by multiplex DNA sequence analysis revealed that mutation fixation at 3, N4-ethenocytosine (varepsilonC) was approximately 3% when the DNA was replicated by normal cell extracts, approximately 48% when replicated by mutA cell extracts and approximately 3% when replicated by mutA recA double mutant cell extracts, in complete agreement with previous in vivo results. Mutagenesis at undamaged DNA sites was significantly elevated by mutA cell-free extracts in the M13 lacZ(alpha) forward mutagenesis system. Neither polA (DNA polymerase I) nor polB (DNA polymerase II) genes are required for the mutA phenotype, suggesting that the phenotype is mediated through a modification of DNA polymerase III or the activation of a previously unidentified DNA polymerase. These findings define the major features of a novel mutagenic pathway and imply the existence of previously unrecognized links between translation, recombination and replication.     

Mutagenesis by aflatoxin in M13 DNA: Base-substitution mechanisms and the origin of strand bias

Summary The two goals of the experiments described here are: (a) to examine whether there is a strand bias in mutagenic processing of bulky lesions in M13 replicative form (RF) DNA, and (b) to examine the mutational mechanisms of metabolically activated aflatoxin. For these experiments, two types of nicked heteroduplex M13 RF DNA molecules (+WT/-am1 and +am1/-WT) in which either the minus (-) or the plus (+) strand carried a gene 1 amber nonsense codon, were constructed. Heteroduplex DNAs were modified in vitro with aflatoxin B₁ activated by hamster liver S9 enzymes, and transfected into SOS(UV)-induced Escherichia coli (Sup^o/uvrA^-/mucAB⁺). Forward mutations in the lacZ -complementing gene segment were scored and sequenced. Results indicated that aflatoxin-induced mutation frequencies in the +WT/-am1 heteroduplex were significantly greater than those in the +am1/-WT heteroduplex, suggesting more efficient mutagenic processing of lesions in the plus strand. These results permit specific suggestions for improved mutation detection in the extensively used M13 forward mutagenesis system. Sequence analysis of point mutations from the +WT/-am1 experiments showed that most substitutions were targeted to plus-strand guanines. Both G-to-A transitions and G-to-T transversions were induced with equal effeciency. Since activated aflatoxin B₁ is known to react almost exclusively with DNA guanines at the N7 position, these results suggest that bulky lesions at guanine N7 position may have the properties of mis-instructional as well as non-instructional lesions.     

Functional recA, lexA, umuD, umuC, polA, and polB genes are not required for the Escherichia coli UVM response.

The Escherichia coli UVM response is a recently described phenomenon in which pretreatment of cells with DNA-damaging agents such as UV or alkylating agents significantly enhances mutation fixation at a model mutagenic lesion (3,N4-ethenocytosine; epsilon C) borne on a transfected M13 single-stranded DNA genome. Since UVM is observed in delta recA cells in which SOS induction should not occur, UVM may represent a novel, SOS-independent, inducible response. Here, we have addressed two specific hypothetical mechanisms for UVM: (i) UVM results from a recA-independent pathway for the induction of SOS genes thought to play a role in induced mutagenesis, and (ii) UVM results from a polymerase switch in which M13 replication in treated cells is carried out by DNA polymerase I (or DNA polymerase II) instead of DNA polymerase III. To address these hypotheses, E. coli cells with known defects in recA, lexA, umuDC, polA, or polB were treated with UV or 1-methyl-3-nitro-1-nitrosoguanidine before transfection of M13 single-stranded DNA bearing a site-specific ethenocytosine lesion. Survival of the transfected DNA was measured as transfection efficiency, and mutagenesis at the epsilon C residue was analyzed by a quantitative multiplex DNA sequencing technology. Our results show that UVM is observable in delta recA cells, in lexA3 (noninducible SOS repressor) cells, in LexA-overproducing cells, and in delta umuDC cells. Furthermore, our data show that UVM induction occurs in the absence of detectable induction of dinD, an SOS gene. These results make it unlikely that UVM results from a recA-independent alternative induction pathway for SOS gene.     

Singlet oxygen mutagenicity induced in the lac operon

We have studied the specificity of singlet oxygen (1O2) mutagenesis in single-stranded DNA phage by analysing 1O2-induced mutations in the lac insert of the M13 mp 19 hybrid phage. 107 lac mutants were analysed showing mainly single-base substitutions with a total of 93% and 7% of 40-50 base deletion mutations. Most of the substitutions are G----T and C----A transversions with respectively 27 and 54% of the mutations. The replicative form of the M13 mp 19 DNA (RFDNA) was used as substrate for the 1O2 reactions, there are then two types of progeny phages DNA's. As guanine residues are the targets of the oxidation, it appears that both types of transversions are provided by one type of lesion: the guanine oxidised by 1O2 is read like a thymine by E. coli DNA polymerase-I.     

Roles of RecA protease and recombinase activities of Escherichia coli in spontaneous and UV-induced mutagenesis and in Weigle repair.

The RecA protein has a second, direct role in the mutagenesis of Escherichia coli and bacteriophage lambda in addition to its first, indirect role of inducing the SOS system by enhancing the proteolytic cleavage of the LexA repressor protein. The need for RecA protease and recombinase functions in the direct role was examined in cells containing split-phenotype RecA mutations, in the absence of LexA protein. Spontaneous mutation of E. coli (his----his+) required both the protease and recombinase activities. The mutation frequency increased with increasing RecA protease strength. In contrast, UV-induced mutation of E. coli required only the RecA protease activity. Weigle repair and mutation of UV-irradiated phage S13 required only RecA protease activity, and even weak activity was highly effective; RecA recombinase activity was not required. RecA+ protein inhibited RecA (Prtc [protease constitutive] Rec+) protein in effecting spontaneous mutation of E. coli. We discuss the nature of the direct role of the RecA protein in spontaneous mutation and in repair and mutagenesis of UV-damaged DNA and also the implications of our results for the theory that SOS-mutable cryptic lesions might be responsible for the enhanced spontaneous mutation in Prtc Rec+ strains.     

Oxidative damage in DNA. Lack of mutagenicity by thymine glycol lesions

Thymine glycol (5,6-dihydroxy-5,6-dihydrothymine) is a base damage common to oxidative mutagens and the major stable radiolysis product of thymine in DNA. We assessed the mutagenic potential of thymine glycols in single-stranded bacteriophage DNA during transfection of Escherichia coli wild-type and umuC strains. cis-Thymine glycols were induced in DNA by reaction with the chemical oxidant, osmium tetroxide (OsO4); modification of thymines was quantitated by using anti-thymine glycol antibody. Inactivation of transfecting molecules showed that one lethal hit corresponded to 1.5 to 2.1 thymine glycols per phage DNA in normal cells, whereas conditions of W-reactivation (SOS induction) reversed 60 to 80% of inactivating events. Forward mutations in the lacI and lacZ' (alpha) genes of f1 and M13 hybrid phage DNAs were induced in OsO4-treated DNA in a dose-dependent manner, in both wild-type and umuC cells. Sequence analysis of hybrid phage mutants revealed that mutations occurred preferentially at cytosine sites rather than thymine sites, indicating that thymine glycols were not the principal pre-mutagenic lesions in the single-stranded DNA. A mutagenic specificity for C----T transitions was confirmed by OsO4-induced reversion of mutant lac phage. Pathways for mutagenesis at derivatives of oxidized cytosine are discussed.