Inducible reactivation of UV-irradiated cholera phage e5 in Vibrio cholerae MAK757

Summary The survival of UV-irradiated cholera phage e5 was found to increase when the host cells, Vibrio cholerae MAK757, were exposed to a low dose of UV irradiation before phage infection (Weigle reactivation), indicating the existence of a UV-inducible DNA repair pathway (SOS repair) in V. cholerae MAK757. The induction signal generated by UV irradiation was transient in nature and lasted about 20–30 min at 37°C. Maximal weigle reactivation of the phage was obtained when the host cells were irradiated with a UV dose of 16 J/m². V. cholerae MAK757 was also found to possess efficient photoreactivation and host cell reactivation of UV-damaged DNA in phage e5.     

Studies on radiation-sensitive mutants of E. coli. 3. Participation of the rec system in induction of mutation by ultraviolet irradiation

Summary The bacterial recA gene participates in the induction by UV irradiation of the clear mutation of phage and the Lac^- mutation of bacteria. The necessary function is induced by irradiation of Rec⁺ bacteria and acts upon DNA irradiated with UV light.     

Kinetics of induction of error-prone repair of bacteriophage lambda by temperature shift in an Escherichia coli dnaB mutant.

Summary Preincubation at 42^o, before infection at permissive temperature by phage , of an Escherichia coli dnaB mutant, provokes a significant increase in survival and mutagenesis of ultraviolet irradiated phage as well as mutagenesis of untreated phage. Similarly to UV irradiation and many chemical mutagens, the inhibition of DNA synthesis by temperature shift of this dnaB mutant induces SOS repair. This work shows that replication blockage in bacterial DNA is not only mutagenic for bacterial DNA itself (Witkin, 1975) but also for normally replicating DNA, probably due to induction of diffusible products.     

Recombinagenic Processing of Uv-Light Photoproducts in Nonreplicating Phage DNA by the Escherichia Coli Methyl-Directed Mismatch Repair System

Nonreplicating lambda phage DNA in homoimmune Escherichia coli lysogens provides a useful model system for study of processes that activate DNA for homologous recombination. We measured recombination by extracting phage DNA from infected cells, using it to transfect recA recipient cells, and scoring the frequency of recombinant infective centers. With unirradiated phage, recombinant frequencies were less than 0.1%. However, recombination could be increased over 300-fold by prior UV irradiation of the phages. The dependence of recombination on UvrA function varied greatly with UV dose. With phage irradiated to 20 J/m2, recombinant frequencies in repressed infections of uvr+ bacteria were one-fifth those in uvrA infections; with phages irradiated to 100 J/m2, frequencies in uvr+ infections were thirty times higher than in uvrA infections. Most UV-stimulated recombination in uvrA infections appeared to depend on the bacterial methyl-directed mismatch-repair system: frequencies were depressed 5-20-fold in uvrA bacteria also lacking MutH, MutL or MutS functions, and recombinant frequencies decreased with increasing GATC-adenine methylation of phage stocks. The biological activity of nonreplicating UV-irradiated phage DNA declined with time after infection of uvrA cells; this decline was photoproduct-dependent, more marked for undermethylated than overmethylated phage DNA, and depended on host MutHLS functions. In uvr+ bacteria, where the UvrABC system provided an alternative, apparently less efficient, route to recombinagenic DNA, UV-stimulated recombinant frequencies were about twice as high in mutH or mutLS as in mut+ cells, in agreement with hyper-rec mut effects previously described by others.     

Ultraviolet reactivation of lambda phage: assay of infectivity of DNA molecules by spheroplast transfection

Prior irradiation of non-lysogenic bacteria by ultraviolet light leads to an increase in the viability of infecting irradiated λ phage (ultraviolet reactivation). Similarly, u.v. irradiation of wild type or uvrD bacteria lysogenic for λcIind^? increased the fraction of closed circular duplex phage DNA molecules formed after infection with u.v.-irradiated λ phage. The closed circular molecules isolated from the irradiated lysogens were shown to be free from u.v. damage by a spheroplast transfection assay. The increase of closed circular molecules is sufficient to explain the ultraviolet reactivation observed by the increase of viability of irradiated phage.In ultraviolet reactivation, damage must be erased on irradiated DNA molecules and the repair is independent of total replication of phage genomes, exchange of sister chromatids or recombination between phage genomes. Protein synthesis is necessary to increase the level of closed circular molecules of irradiated λ phage after irradiation of bacteria.     

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.     

DNA repair in Bacillus subtilis. I. The presence of an inducible system.

Summary Following UV irradiation of Bacillus subtilis there is a coordinate induction of: 1) a new protein, 2) a W-reactivation system, 3) a DNA modification system, and 4) prophages. These functions are induced following UV irradiation of repair proficient bacteria and mutants deficient in excision repair (uvr-1) and DNA polymerase I activity (polA5). However, they are not induced, or are impaired in their ability to be induced in bacteria containing the recA1 and the recG13 mutations. This inducible system is compared to the SOS system observed in E. coli.     

DNA Structures Generated during Recombination Initiated by Mismatch Repair of Uv-Irradiated Nonreplicating Phage DNA in Escherichia Coli: Requirements for Helicase, Exonucleases, and Recf and Recbcd Functions

During infection of homoimmune Escherichia coli lysogens (``repressed infections'), undamaged non-replicating λ phage DNA circles undergo very little recombination. Prior UV irradiation of phages dramatically elevates recombinant frequencies, even in bacteria deficient in UvrABC-mediated excision repair. We previously reported that 80-90% of this UvrABC-independent recombination required MutHLS function and unmethylated d(GATC) sites, two hallmarks of methyl-directed mismatch repair. We now find that deficiencies in other mismatch-repair activities--UvrD helicase, exonuclease I, exonuclease VII, RecJ exonuclease--drastically reduce recombination. These effects of exonuclease deficiencies on recombination are greater than previously observed effects on mispair-provoked excision in vitro. This suggests that the exonucleases also play other roles in generation and processing of recombinagenic DNA structures. Even though dsDNA breaks are thought to be highly recombinagenic, 60% of intracellular UV-irradiated phage DNA extracted from bacteria in which recombination is low--UvrD(-), ExoI(-), ExoVII(-), or RecJ(-)--displays (near-)blunt-ended dsDNA ends (RecBCD-sensitive when deproteinized). In contrast, only bacteria showing high recombination (Mut(+) UvrD(+) Exo(+)) generate single-stranded regions in nonreplicating UV-irradiated DNA. Both recF and recB recC mutations strikingly reduce recombination (almost as much as a recF recB recC triple mutation), suggesting critical requirements for both RecF and RecBCD activity. The mismatch repair system may thus process UV-irradiated DNA so as to initiate more than one recombination pathway.     

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.     

Photodynamic effect of proflavine on ?X174 bacteriophage, its DNA replicative form and its isolated single-stranded DNA: Inactivation, mutagenesis and repair

Summary In contrast to that what is observed with most inactivating agents, proflavine-mediated photoinactivation is about 10 times more efficient on double-stranded X 174 replicative form DNA (RFI) than on isolated single-stranded X 174 DNA. Both X RFI DNA and encapsidated DNA have similar sensitivities to proflavine and light treatment.With the three substrates studied, reactivation can occur through high multiplicity of infection and depends upon the cellular rec A gene product. No effect of the pol A, uvr A or lex A gene mutations has been found on either phage or DNA inactivation rates.The photodynamically induced lesions can be repaired at least in part, by the SOS repair system induced in the host-cells by a 100 J ·m^-2 UV irradiation. SOS repair does not occur with bacteria (or spheroplasts) irradiated in the presence of chloramphenicol.Reversion frequency of the X 174 amber mutations indicates that 1) photodynamically induced lesions are mutagenic whether the rec A gene product is present or not in the indicator bacteria; 2) induction of the SOS repair system is accompanied by a mutagenic process which almost results in a two fold increase of the reversion frequency; and 3) multiplicity reactivation occurs through a recombinational process and is not mutagenic per se.     

Repair and recombination of nonreplicating UV-irradiated phage DNA in E. coli II. Stimulation of RecF-dependent recombination by excision repair of cyclobutane pyrimidine dimers and of other photoproducts

Summary Three aspects of recombination of UV-irradiated nonreplicating lambda phage DNA were addressed: the photoproduct(s) responsible, the role of UvrABC-mediated excision repair, and the dependence on RecF function.Cyclobutane pyrimidine dimers appeared responsible for some recombination because photoreactivation reduced the frequency of 254-nm-stimulated recombination and because photosensitized 313-nm irradiation stimulated recombination. Other photoproducts seemed recombinogenic as well, because high fluences of 254-nm irradiation stimulated recombination considerably more, per cyclobutane dimer induced, than photosensitized 313-nm irradiation, and because photoreactivation did not eliminate 254-nm stimulated recombination. For both treatments, much, but not all, of the recombination was UvrABC-dependent. Recombination was mostly RecF-dependent, but was not affected by recB recC or recE mutationsThe first paper in this series is Hays et al., (1985)     

Weigle reactivation of the single-stranded DNA phage f1

Summary The survival of ultraviolet light (UV) damaged single-stranded DNA bacteriophage f1 is increased when the Escherichia coli host is irradiated with UV prior to infection. This repair, called Weigle reactivation, is multiplicity independent and is absent in recA and in lexA mutants. The function of the recA-lexA repair system needed is repair and not recombination, as demonstrated by the absence of Weigle reactivation in mutants that are recombination proficient but defective in repair of double-stranded DNA. Weigle reactivation of f1 requires high levels of the recA protein, and in addition activation of recA or another protein. This activation can be produced by UV irradiation, or by the tif-1 allele of recA together with the spr allele of lexA. Mutagenesis of f1 has the same requirements as W-reactivation, and in addition requires UV irradiation of the phage.     

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.     

Stimulation of recombination between homologous sequences on carcinogen-treated plasmid DNA and chromosomal DNA by induction of the SOS response in Escherichia coli K12

Summary Previous studies have shown that transformation of Escherichia coli by plasmid DNA modified in vitro by carcinogens leads to RecA-dependant recombination between homologous plasmid and chromosomal DNA sequences. The mechanism of this recombination has now been studied using recombination-deficient mutants, and the influence of induction of the SOS response on the level of recombination investigated. Plasmid pNO1523, containing the str ⁺ operon (Sm^s), has been modified in vitro by either irradiation with UV light, or by reaction with (±) trans-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) and used to transform streptomycin-resistant hosts. The formation of Amp^r transformants which also carry streptomycin resistance was used as a measure of the level of recombination between plasmid and chromosomal DNA. Transformation of recB and recC mutants produced no change in the level of recombination while in the recF mutant a significant decrease was observed compared to the wild type host. Thermal induction of the SOS response in tif-1 and tif-1 umuC mutants followed by transformation led to a four-fold increase in recombination in both cases. The results suggest that the streptomycin-resistant transformants arise exclusively via a recombinational pathway which is largely dependant on the recF gene product, and that this pathway is influenced by induction of the SOS response. These results are discussed in terms of the mechanism of this recombination.     

Role of the bacterial and phage recombination systems and of DNA replication in genetic recombination of UV-irradiated phage lambda

Summary In this paper are studied in E. coli K12 the influence of the bacterial Rec and phage Red recombination systems on the rescue of the O ⁺ gene from the prophage by a superinfecting O ^- phage, UV irradiated or not. In the absence of UV irradiation the Red system produces more recombinants that does the Rec system, and its action requires DNA replication. The presence of UV lesions in the DNA facilitates the action of the Rec system, which is more efficient in this instance than the Red system and can act in the absence of DNA replication. In all cases, there is a cooperation between the two generalized recombination systems.     

Bacteriophage T4 DNA polymerase determines the amount and specificity of ultraviolet mutagenesis

Summary Ultraviolet mutagenesis in bacteriophage T4 proceeds via error-prone repair (EPR) and requires the functional integrity of the uvsWXY system which mediates genetic recombination, recombinational repair, and mutability by diverse DNA damaging agents. Current opinion holds that mutagens acting through EPR generate DNA damage which blocks the progress of the replication complex and that EPR consists of the facilitated bypass of such inaccurate, damaged templates. This notion predicts that the T4 DNA polymerase (encoded by gene 43) mediates EPR in UV irradiated phage T4. This prediction is verified by the discovery that gene 43 mutations often enhance or reduce UV mutagenesis (which is scored by the induction of r mutants) and sometimes change its specificity.     

SOS repair of 8-methoxypsoralene monoadducts in DNA of lambda bacteriophage and plasmids is mediated by MucA 2 ′ B, but not UmuD 2 ′ C (PolV) polymerase

The light-induced action of 8-methoxypsoralen (8-MOP) on λ phage and plasmids yields monoadducts and interstrand crosslinks. The survival and clear plaque mutation frequency in the phage photosensitized with 8-MOP and irradiated with UV at wavelength >320 nm are increased when the wild-type host (Escherichia coli uvr ⁺) is subjected to UV irradiation (wavelength = 254 nm) prior to phage inoculation. These phenomena are known as “W reactivation” and “W mutagenesis.” It is shown that 8-MOP monoadducts in λ DNA induce clear mutations in the phage inoculated to UV-irradiated excision repair mutants of E. coli only when the error-prone repair is performed by MucA ₂ ^′ B, but not PolV (UmuD ₂ ^′ C) polymerase. The efficiency of the SOS repair (W reactivation) of 8-MOP monoadducts in plasmid and λ phage DNA also only increases with the presence of pKM101 plasmid muc ⁺ in E. coli uvr ^?.     

Mutagenic DNA repair in Escherichia coli. VI. Gamma radiation mutagenesis in a tif-1 strain.

Summary In the non-filamenting tif-1 strain WP44_s NF trp a dramatic enhancement of both UV and gamma ray mutability to Trp⁺ was observed when irradiated bacteria were incubated on plates at 43°. This enhanced mutability was progressively suppressed when the initial plating density exceeded 10⁸ bacteria per plate and was not demonstrable in liquid media. Under optimal conditions more mutants were induced by gamma radiation than could reasonably be accounted for by the initial number of radiation-induced lesions in the DNA, implying the existence of some mechanism for amplifying the radiation effect. Moreover, the tif-enhanced mutation frequency could be obtained if incubation at restrictive temperature was delayed for up to 60 min in nutrient broth after irradiation, at a time when all known reparable DNA damage had been repaired and the number of viable bacteria had more than doubled. On plates the effect of high temperature was still fully demonstrable 120 min after irradiation. The results are hard to reconcile with the hypothesis that incubation of tif-1 bacteria at restrictive temperature causes the induction of a repair system acting on DNA damaged by gamma radiation. A more compatible interpretation would be that radiation causes a persisting physiological disturbance in the cell and that this enhances the spontaneous mutator effect occurring in tif-1 bacteria subjected to subsequent thermal shock.     

Suppression of Escherichia coli recF mutations by recA-linked srfA mutations.

Suppressors of recF (srfA) were found by selection for resistance to mitomycin C and UV irradiation in a recB21 recC22 sbcB15 recF143 strain. srfA mutations map in recA and are dominant to srfA+. They suppress both the DNA repair and the recombination deficiencies due to recF mutations. Therefore, RecA protein which is altered by the srfA mutation can allow genetic recombination to proceed in the absence of recB, recC, and recF functions. recF is also required for induction of the SOS response after UV damage. We propose that recF+ normally functions to allow the expression of two recA activities, one that is required for the RecF pathway of recombination and another that is required for SOS induction. The two RecA activities are different and are separable by mutation since srfA mutations permit recombination to proceed but have not caused a dramatic increase in SOS induction in recF mutants. According to this hypothesis, one role for recF in DNA repair and recombination is to modulate RecA activities to allow RecA to participate in these recF-dependent processes.     

Involvement of alkylhydroxybenzenes in the <Emphasis Type="Italic">Escherichia coli</Emphasis> response to the lethal effect of UV irradiation

This work is concerned with the role of alkylhydroxybenzenes (AHBs), chemical analogs of the autoregulatory microbial d ₁ factors, on the development of the stress response of bacterial cells to UV irradiation, including SOS system induction, preservation of cell viability, and S → R phase transitions of the Escherichia coli test strain with the bioluminescence genes cloned under the control of the recA gene promoter. UV irradiation, a natural stress factor, and an increase in AHB concentrations were found to elicit uniform responses in bacteria, indicating that AHBs function as alarmones, i.e., alarm signals. It was revealed that preincubating bacteria with alkylhydroxybenzenes considerably enhanced their viability upon irradiation with lethal UV doses; this was accompanied by a relative decrease in the SOS response activity and a concomitant increase in the frequency of phase transitions. The efficiency of the protective action of AHBs increased with an increase in their hydrophobicity degree. The probable mechanism of the protective effect of AHBs is discussed, based on their capacity for the interaction with biopolymers, which results in changing their structural organization and conferring resistance to a broad spectrum of stress factors. Such a “passive” protective mechanism reduces the susceptibility of DNA to UV irradiation, causing a decrease in the parameters related to the SOS system induction that is responsible for the “active” protective mechanism in bacterial cells. As a result, viability retention under the lethal influence of UV irradiation is possible at minimal values of repair activity and is accompanied by an increase in the phenotypic variability of the surviving part of a bacterial population.