Survival and induction of SOS in Escherichia coli treated with cisplatin, UV-irradiation, or mitomycin C are dependent on the function of the RecBC and RecFOR pathways of homologous recombination

Resistance of tumors to drugs such as cisplatin and mitomycin C (MMC) is an important factor limiting their usefulness in cancer chemotherapy. The antitumor effects of these drugs are due to the formation of bifunctional adducts in DNA, with cisplatin causing predominantly intrastrand-crosslinks and MMC causing interstrand-crosslinks. The SOS chromotest was used to study the cellular mechanisms that process DNA damage in Escherichia coli exposed to cisplatin, ultraviolet irradiation (UV) and MMC and subsequently facilitate the production of a molecular signal for induction of the SOS response. Strains used in the SOS chromotest have a fusion of lacZ with the sfiA (sulA) gene so that the amount of SOS inducing signal, which is modulated by the ability of the cell to repair DNA, is measured by assaying beta-galactosidase activity. SOS induction in a strain proficient in homologous recombination (HR) was compared with that in isogenic strains deficient in HR due to a blocked RecBC pathway caused by a recB mutation or a blocked RecFOR pathway caused by a recO mutation. The effect of cisplatin treatment in a uvrA mutant strain blocked at the first step of NER was compared with that in an isogenic strain proficient in NER. Cellular resistance was measured as percent colony forming units (cfu) for cells treated with increasing doses of cisplatin, MMC and UV relative to that in untreated control cultures. The importance of both HR pathways for resistance to these treatments was demonstrated by decreased survival in mutants with the recB mutant being more sensitive than the recO mutant. SOS induction levels were elevated in the sensitive recB strain relative to the HR proficient strain possibly due to stalled and/or distorted replication forks at crosslinks in DNA. In contrast, induction of SOS was dependent on RecFOR activity that is thought to act at daughter strand gaps in newly synthesized DNA to mediate production of the signal for SOS induction. Proficiency in NER was necessary for both survival and high levels of SOS induction in cisplatin treated cells.     

Analysis of the SOS inducing signal in Bacillus subtilis using Escherichia coli LexA as a probe.

We analyzed the Bacillus subtilis SOS response using Escherichia coli LexA protein as a probe to measure the kinetics of SOS activation and DNA repair in wild-type and DNA repair-deficient strains. By examining the effects of DNA-damaging agents that produce the SOS inducing signal in E. coli by three distinct pathways, we obtained evidence that the nature of the SOS inducing signal has been conserved in B. subtilis. In particular, we used the B. subtilis DNA polymerase III inhibitor, 6-(p-hydroxyphenylazo)-uracil, to show that DNA replication is required to generate the SOS inducing signal following UV irradiation. We also present evidence that single-stranded gaps, generated by excision repair, serve as part of the UV inducing signal. By assaying the SOS response in B. subtilis dinA, dinB, and dinC mutants, we identified distinct deficiencies in SOS activation and DNA repair that suggest roles for the corresponding gene products in the SOS response.     

UV induction of LexA independent proteins which could be involved in SOS repair

The SOS response is induced in E coli following treatments that interfere with DNA replication. The response is under the control of the recA and the lexA genes. Strains defective in LexA repressor constitutively express SOS proteins. However, SOS repair does not reach its maximum level in these strains. Instead, an activation of RecA protein and de novo protein synthesis are required for full repair. We have analyzed by 2-dimensional gel electrophoresis the induction of proteins after UV irradiation of lexA(Def) bacteria. Proteins which might participate in SOS repair are induced under these conditions.     

The effect of am umuC mutation on the induction of an SOS response in E. coli cella under the action of UV and gamma irradiation

The kinetics of the SOS induction in E. coli cells of wild type and deficient in umuC gene exposed to UV and gamma-rays were analysed. In the presence of UmuC protein SOS induction was 3-5.5 times lower and delayed for about 30 minutes after both UV and gamma rays. It was shown that decrease of the SOS induction in wild type cells irradiated by UV was due to more effective elimination of the photolesions from DNA by excision repair system. UmuCD-dependent inhibition of DNA replication was discussed as a possible mechanism allowing additional time for error-free repair.     

Mutagenic DNA repair in Escherichia coli XXI. A stable SOS-inducing signal persisting after excision repair of ultraviolet damage

Mutations to streptomycin resistance induced by ultraviolet light in Escherichia coli can lose their susceptibility to photoreversing light during excision repair and in the absence of chromosomal replication and protein synthesis, i.e., under conditions where SOS induction cannot occur. Using fusions of lac with sulA and umuC we have shown that after excision of UV damage in the presence of chloramphenicol there is a persisting, relatively stable signal capable of inducing SOS genes when protein sysnthesis is subsequently permitted. The persisting signal is formed roughly in proportion to the square of the UV dose and is about 30% photoreversible. It is suggested that the persisting SOS-inducing signal comprises a UV photoproduct (the target lesion) opposite a gap in the opposing DNA strand, and is formed by excision of one (the ancillary lesion) of a pair of closely opposed photoproducts. Calculations suggest that as few as two or three such configurations in a cell can lead to induction a sulA when protein synthesis is permitted. It is not clear whether these configurations can directly induce the SOS system because of their region of single-stranded DNA or whether the ultimate SOS-inducing signal is a more extensive single-stranded region formed when such configurations encounter a replication fork. Photoproduct/gap configurations have been previously suggested to be potentially mutagenic. UV-induced mutations to streptomycin resistance are mostly at A:T sites and are not photoreversible in fully SOS-induced bacteria in the absence of excision repair, indicating that they are not targeted at cyclobutane-type pyrimidine dimers. In SOS-induced excision-proficient bacteria there is about 39% photoreversibility which is rapidly lost after UV. This photoreversibility is attributed to many ancillary lesions being cyclobutane-type pyrimidine dimers which are excised leading to the exposure of target lesions on the opposing strand which, at these particular sites, are mostly non-photoreversible photoproducts.     

Mutagenic DNA repair in Escherichia coli. XXI. A stable SOS-inducing signal persisting after excision repair of ultraviolet damage.

Mutations to streptomycin resistance induced by ultraviolet light in Escherichia coli can lose their susceptibility to photoreversing light during excision repair and in the absence of chromosomal replication and protein synthesis, i.e., under conditions where SOS induction cannot occur. Using fusions of lac with sulA and umuC we have shown that after excision of UV damage in the presence of chloramphenicol there is a persisting, relatively stable signal capable of inducing SOS genes when protein sysnthesis is subsequently permitted. The persisting signal is formed roughly in proportion to the square of the UV dose and is about 30% photoreversible. It is suggested that the persisting SOS-inducing signal comprises a UV photoproduct (the target lesion) opposite a gap in the opposing DNA strand, and is formed by excision of one (the ancillary lesion) of a pair of closely opposed photoproducts. Calculations suggest that as few as two or three such configurations in a cell can lead to induction a sulA when protein synthesis is permitted. It is not clear whether these configurations can directly induce the SOS system because of their region of single-stranded DNA or whether the ultimate SOS-inducing signal is a more extensive single-stranded region formed when such configurations encounter a replication fork. Photoproduct/gap configurations have been previously suggested to be potentially mutagenic. UV-induced mutations to streptomycin resistance are mostly at A:T sites and are not photoreversible in fully SOS-induced bacteria in the absence of excision repair, indicating that they are not targeted at cyclobutane-type pyrimidine dimers. In SOS-induced excision-proficient bacteria there is about 39% photoreversibility which is rapidly lost after UV. This photoreversibility is attributed to many ancillary lesions being cyclobutane-type pyrimidine dimers which are excised leading to the exposure of target lesions on the opposing strand which, at these particular sites, are mostly non-photoreversible photoproducts.     

Nature of the SOS-inducing signal in Escherichia coli. The involvement of DNA replication

The SOS genes of Escherichia coli, which include many DNA repair genes, are induced by DNA damage. Although the central biochemical event in induction, activation of RecA protein through binding of single-stranded DNA and ATP to promote cleavage of the LexA repressor, is known, the cellular event that provides this activation following DNA damage has not been well understood. We provide evidence here that the major pathway of induction after damage by a typical agent, ultraviolet light, requires an active replication fork; this result supports the model that DNA replication leaves gaps where elongation stops at damage-induced lesions, and thus provides the single-stranded DNA that activates RecA protein. In order to detect quantitatively the immediate product of the inducing signal, activated RecA protein, we have designed an assay to measure the rate of disappearance of intact LexA repressor. With this assay, we have studied the early phase of the induction process. LexA cleavage is detectable within minutes after DNA damage and occurs in the absence of protein synthesis. By following the reaccumulation of LexA in the cell, we detect repair of DNA and the disappearance of the inducing signal. Using this assay, we have measured the LexA content of wild-type and various mutant cells, characterized the kinetics and conditions for development of the inducing signal after various inducing treatments and, finally, have shown the requirement for DNA replication in SOS induction by ultraviolet light.     

Induction by UV light of the SOS function sfiA in Escherichia coli strains deficient or proficient in excision repair.

The influence of the nucleotide excision repair system on the induction by UV irradiation of the SOS function sfiA has been investigated. The level of sfiA expression was monitored by means of a sfiA::lacZ operon fusion in both the wild-type strain and a uvrA mutant. We found that the initial steady rate of sfiA expression was proportional to the UV dose and was identical in uvr+ and uvrA backgrounds. This suggests that the initial steady rate of sfiA expression is determined by the initial number of lesions and before any effect of excision repair. We confirmed that after 2 h of expression the net synthesis of sfiA product is, for the same UV dose, about five times lower in uvr+ than in uvrA strains. We show that this is due to earlier repression of the SOS system in uvr+ than in uvrA strains and not to different initial rates.     

DNA repair and its relation to cell division in E. coli

In E. coli, lesions introduced by agents such as UV radiation, chemical agents, thymine starvation, lead to the induction of a series of bacterial fonctions called the "SOS response" (DNA reparation, mutagenesis, filamentation, prophages induction etc...). Genetics and biochemical study set up the evidences of a regulated mechanism. The central effector of this mechanism is the Rec A protéin. When a cell's DNA is damaged or its DNA replication is inhibited, an inducing signal is generated. The inducing signal reversebly activates a specific protease activity of Rec A which allows it to cleave the Lex A repressor. Thus inducing operons repressed by Lex A. These operons are implicated in DNA reparations and also in cellular division. A coordination between reparation and cellular division is thus established.     

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.     

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.     

Effect of the adaptive response on the induction of the SOS pathway in E. coli K-12

Summary The adaptive-response is an inducible repair system of E. coli which reduces the mutagenic and cytotoxic effects of alkylation damage (Samson and Cairns, 1977). In adapted cells (cells exposed to sublethal doses of alkylating agents) the induction of W-reactivation and W-mutagenesis by alkylating agents is almost totally blocked. Despite the fact that adaptation has no detectable effect on UV mutagenesis in E. coli K-12, it does inhibit to some extent the UV and tif-1 mediated induction of SOS functions such as W-reactivation and prophage induction. Furthermore, the kinetics of induction of W-mutagenesis following UV treatment are altered by adaptation. In this case the adaptive-response seems to specifically block the induction of an error-producing W-reactivating capacity which normally would increase soon after UV treatment, while affecting error-free W-reactivating systems to a lesser extent.     

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.     

Temporal control of colicin E1 induction.

The expression of the gene encoding colicin E1, cea, was studied in Escherichia coli by using cea-lacZ gene fusions. Expression of the fusions showed the same characteristics as those of the wild-type cea gene: induction by treatments that damage DNA and regulation by the SOS response, sensitivity to catabolite repression, and a low basal level of expression, despite the presence of the fusion in a multicopy plasmid. Induction of expression by DNA-damaging treatments was found to differ from other genes involved in the SOS response (exemplified by recA), in that higher levels of DNA damage were required and expression occurred only after a pronounced delay. The delay in expression following an inducing treatment was more pronounced under conditions of catabolite repression, indicating that the cyclic AMP-cyclic AMP receptor protein complex may play a role in induction. These observations also suggest a biological rationale for the control of cea expression by the SOS response and the cyclic AMP-cyclic AMP receptor protein catabolite repression system.     

Regulation of the Escherichia coli K-12 uvrB operon

The UV light inducibility of the uvrB operon of Escherichia coli K-12 was previously demonstrated by exploiting a strain in which the gene for the enzyme beta-galactosidase was inserted into the uvrB operon. This insert is now shown to be located within the structural gene for the uvrB enzyme, leaving the regulatory sequences of the operon intact. Analyses to quantitate the induction of this system show that derepression of the operon is first detectable 5 min after UV exposure, with the rate of synthesis increasing to four to six times the uninduced rate during the subsequent 30 min. Induction is unaffected by mutations in other components of nucleotide excision repair. The control of uvrB was found to result from direct repression by the lexA gene product, with the recA gene product playing an indirect role. Nucleotide excision repair thus seems to be part of the SOS response.     

Detection of ionizing radiations with the SOS Chromotest, a bacterial short-term test for genotoxic agents

The effects of 3 types of ionizing radiation, gamma-rays, neutrons and accelerated alpha-particles, were examined using the SOS Chromotest, a bacterial colorimetric assay for genotoxic agents based on the measurement of the SOS response in Escherichia coli. The SOS Chromotest appeared to be a sensitive and simple assay to detect quantitatively these radiations as well as their biological effects. The range of adsorbed doses for which induction was observed was similar for the 3 types of radiation, the minimum inducing doses being in the order of 2.5-5 Gy. We discuss the possible use of these observations to study the molecular action of radiations and to compare their genotoxic effects with those of chemicals.     

The universal stress protein A of Escherichia coli is required for resistance to DNA damaging agents and is regulated by a RecA/FtsK-dependent regulatory pathway

The link between cell division defects and the induction of the universal stress response is demonstrated to operate via the RecA regulator of the SOS response. An insertion in the cell division gene ftsK upregulates uspA in a recA-dependent manner. Unlike true SOS response genes, this upregulation only occurs in growth-arrested cells and is LexA independent. Thus, besides ppGpp-dependent starvation signals, DNA aberrations transduce RecA-dependent signals to the uspA promoter, which only affect the promoter during stasis. Further, we show that ftsK itself, like uspA, is induced in stationary phase and that this induction requires the stringent control modulon rather than activated RecA. Thus, ftsK, like uspA, is regulated by at least two global regulators: ppGpp of the stringent control network and RecA of the SOS modulon. We suggest that UspA is a new bona fide member of the RecA-dependent DNA protection and repair system, as mutants lacking functional UspA were found to be sensitive to UV irradiation and mitomycin C exposure. Moreover, the UV sensitivity of uspA mutants is enhanced in an additive manner by the ftsK1 mutation.