Repair of damage in double-stranded phi X174 (RF) DNA due to radiation-induced water radicals

Experiments in which the yields of radiation-induced OH and H radicals were varied, showed that both types of water radicals inactivate phi X174 RF DNA to about the same extent as measured by transfection of the (irradiated) DNA to E. coli wild-type spheroplasts. On the other hand, using spheroplasts prepared from E. coli strains, deficient in one of the proteins involved in excision DNA repair (uvrA- or uvrC-) or in post-replication repair (recA-), clear differences between damage originating from OH or H radical attack were found. Part of the radiation damage due to H radicals appeared to be repairable by an uvrA-gene-dependent repair mechanism, whereas this repair pathway does not play an important role in the case of OH radical damage. The reverse applies to uvrC-gene-dependent repair, which only affects OH radical damage (obtained under anoxic conditions), but has no influence on damage due to H radicals. Irradiation of double-stranded phi X174 (RF) DNA in the presence of oxygen however, yields damage--due to OH radicals only--which appeared not to be sensitive to either uvrC- or uvrA-gene-dependent repair. Furthermore, post-replication repair (recA) has only very little effect on the amount of inactivation by H or OH radicals, when irradiation is carried out under anoxic conditions. We did not find significant inactivation due to hydrated electrons, whether the biological activity was determined by use of wild-type spheroplasts or of strains deficient in excision or post-replication repair proteins.     

Efficient repair of hydrogen peroxide-induced DNA damage by Escherichia coli requires SOS induction of RecA and RuvA proteins

The survival of Escherichia coli following treatment with a low dose (1-3 mM) of hydrogen peroxide (H(2)O(2)) that causes extensive mode-one killing of DNA repair mutants is stimulated by the induction of the SOS regulon. Results for various mutants indicate that induction of recA and RecA protein-mediated recombination are critical factors contributing to the repair of H(2)O(2)-induced oxidative DNA damage. However, because DNA damage activates RecA protein's coprotease activity essential to cleavage of LexA repressor protein and derepression of all SOS genes, it is unclear to what extent induction of RecA protein stimulates this repair. To make this determination, we examined mode-one killing of DeltarecA cells carrying plasmid-borne recA (P(tac)-recA(+)) and constitutively expressing a fully induced level of wild-type RecA protein when SOS genes other than recA are non-inducible in a lexA3 (Ind(-)) genetic background or inducible in a lexA(+) background. At a H(2)O(2) dose resulting in maximal killing, DeltarecA lexA3 (Ind(-)) cells with P(tac)-recA(+) show 40-fold greater survival than lexA3 (Ind(-)) cells with chromosomal recA having a low, non-induced level of RecA protein. However, they still show 10- to 15-fold lower survival than wild-type cells and DeltarecA lexA(+) cells with P(tac)-recA(+). To determine if the inducible RuvA protein stimulates survival, we examined a ruvA60 mutant that is defective for the repair of UV-induced DNA damage. This mutant also shows 10- to 15-fold lower survival than wild-type cells. We conclude that while induction of RecA protein has a pronounced stimulatory effect on the recombinational repair of H(2)O(2)-induced oxidative DNA damage, the induction of other SOS proteins such as RuvA is essential for wild-type repair.     

Free radical scavenging and the expression of potentially lethal damage in X-irradiated repair-deficient Escherichia coli

When cells are exposed to ionizing radiation, they suffer lethal damage (LD), potentially lethal damage (PLD), and sublethal damage (SLD). All three forms of damage may be caused by direct or indirect radiation action or by the interaction of indirect radiation products with direct DNA damage. In this report I examine the expression of LD and PLD caused by the indirect action of X rays in isogenic, repair-deficient Escherichia coli. The radiosensitivity of a recA mutant, deficient both in pre- and post replication recombination repair and SOS induction (inducible error-prone repair), was compared to that of a recB mutant which is recombination deficient but SOS proficient and to a previously studied DNA polymerase 1-deficient mutant (polA) which lacks the excision repair pathway. Indirect damage by water radicals (primarily OH radicals) was circumvented by the presence of 2 M glycerol during irradiation. Indirect X-ray damage by water radicals accounts for at least 85% of the PLD found in exposed repair-deficient cells. The DNA polymerase 1-deficient mutant is most sensitive to indirect damage with the order of sensitivity polA1 greater than recB greater than or equal to recA greater than wild type. For the direct effects of X rays the order of sensitivity is recA greater than recB greater than polA1 greater than wild type. The significance of the various repair pathways in mitigating PLD by direct and indirect damage is discussed.     

细菌DNA损伤修复的诱导、调控及结局的研究进展

DNA损伤修复(SOS反应)是细菌适应环境、抵抗外界压力和修复自身损伤的重要机制.为了解SOS反应的过程,全面揭示细菌生存机制,本研究对DNA损伤修复的过程、调节及适应性变化进行文献综述.结果 表明,内源和外源的诸多压力都可以激活SOS反应,抗生素是激活该反应的主要因素.RecA在感知外界压力和系统启动过程中发挥重要作...     

Effect of cordycepin(3'-deoxyadenosine) on excision repair of 5,6-dihydroxy-dihydrothymine-type products from the DNA of Micrococcus radiodurans

Cordycepin(3'-deoxyadenosine), a nucleoside analog, has been shown to enhance radiation-induced cell killing. In an effort to elucidate the possible mechanism for enhancement of cell killing, the effect of cordycepin on the excision repair of radiation-induced 5,6-dihydroxy-dihydrothymine-type (t') products from the DNA of wild type Micrococcus radiodurans was investigated. The capacity of M. radiodurans to excise nondimeric (t') products from its DNA was significantly impaired after cordycepin treatment. The results suggest that the increased radiation sensitivity of cordycepin-treated cells could be due to alterations in cellular processes that repair DNA damage.     

Inducible SOS response system of DNA repair and mutagenesis in Escherichia coli

Chromosomal DNA is exposed to continuous damage and repair. Cells contain a number of proteins and specific DNA repair systems that help maintain its correct structure. The SOS response was the first DNA repair system described in Escherichia coli induced upon treatment of bacteria with DNA damaging agents arrest DNA replication and cell division. Induction of the SOS response involves more than forty independent SOS genes, most of which encode proteins engaged in protection, repair, replication, mutagenesis and metabolism of DNA. Under normal growth conditions the SOS genes are expressed at a basal level, which increases distinctly upon induction of the SOS response. The SOS-response has been found in many bacterial species (e.g., Salmonella typhimurium, Caulobacter crescentus, Mycobacterium tuberculosis), but not in eukaryotic cells. However, species from all kingdoms contain some SOS-like proteins taking part in DNA repair that exhibit amino acid homology and enzymatic activities related to those found in E. coli. but are not organized in an SOS system. This paper presents a brief up-to-date review describing the discovery of the SOS system, the physiology of SOS induction, methods for its determination, and the role of some SOS-induced genes.     

SOS regulatory elements are essential for UPEC pathogenesis

Epithelial cells are highly regarded as the first line of defense against microorganisms, but the mechanisms used to control bacterial diseases are poorly understood. A component of the DNA damage repair regulon, SulA, is essential for UPEC virulence in a mouse model for human urinary tract infection, suggesting that DNA damage is a key mediator in the primary control of pathogens within the epithelium. In this study, we examine the role of DNA damage repair regulators in the intracellular lifestyle of UPEC within superficial bladder epithelial cells. LexA and RecA coordinate various operons for repair of DNA damage due to exogenous and endogenous agents and are known regulators of sulA. UPEC strains defective in regulation of the SOS response mediated by RecA and LexA display attenuated virulence in immunocompetent mice within the first 6 h post infection. RecA and LexA regulation of the SOS regulon is dispensable in immunocompromised mice. These data suggest that epithelial cells produce sufficient levels of DNA damaging agents, such that the bacterial DNA damage repair response is essential, as a means to control invading bacteria. Since many pathogens interact with the epithelium before exposure to professional phagocytes, it is likely that adaptation to oxidative radicals during intracellular growth provides additional protection from killing by innate immune phagocytes.     

Increase in plasmid transformation efficiency in SOS-induced Escherichia coli cells

Summary UV irradiation of competent cells of Escherichia coli K12 produced an increase in the efficiency of transformation with plasmid DNA. This phenomenon has been called IPTE (increase in plasmid transformation efficiency) and is dependent on the activated state of the RecA protein. IPTE is independent of the lexA, recB recC, and recF genes. It is not related to the size or the replicon type of the plasmid. Furthermore, it is also induced in cells which have been previously treated with other SOS system-inducing agents such as bleomycin, mitomycin C, or nalidixic acid. IPTE is therefore similar to other repair (SOS) functions inducible by DNA damage since all of them are dependent upon activation of the RecA protein. IPTE differs from other SOS functions in the absence of a direct control by the LexA repressor.     

Recombinational repair is critical for survival of Escherichia coli exposed to nitric oxide

Nitric oxide (NO(.)) is critical to numerous biological processes, including signal transduction and macrophage-mediated immunity. In this study, we have explored the biological effects of NO(.)-induced DNA damage on Escherichia coli. The relative importance of base excision repair, nucleotide excision repair (NER), and recombinational repair in preventing NO(.)-induced toxicity was determined. E. coli strains lacking either NER or DNA glycosylases (including those that repair alkylation damage [alkA tag strain], oxidative damage [fpg nei nth strain], and deaminated cytosine [ung strain]) showed essentially wild-type levels of NO(.) resistance. However, apyrimidinic/apurinic (AP) endonuclease-deficient cells (xth nfo strain) were very sensitive to killing by NO(.), which indicates that normal processing of abasic sites is critical for defense against NO(.). In addition, recA mutant cells were exquisitely sensitive to NO(.)-induced killing. Both SOS-deficient (lexA3) and Holliday junction resolvase-deficient (ruvC) cells were very sensitive to NO(.), indicating that both SOS and recombinational repair play important roles in defense against NO(.). Furthermore, strains specifically lacking double-strand end repair (recBCD strains) were very sensitive to NO(.), which suggests that NO(.) exposure leads to the formation of double-strand ends. One consequence of these double-strand ends is that NO(.) induces homologous recombination at a genetically engineered substrate. Taken together, it is now clear that, in addition to the known point mutagenic effects of NO(.), it is also important to consider recombination events among the spectrum of genetic changes that NO(. ) can induce. Furthermore, the importance of recombinational repair for cellular survival of NO(.) exposure reveals a potential susceptibility factor for invading microbes.     

Inducible DNA repair and differentiation in Bacillus subtilis: interactions between global regulons

The SOS response of Escherichia coli has become a paradigm for the study of inducible DNA repair and recombination processes in many different organisms. While these studies have demonstrated that the components of the SOS response appear to be highly conserved among bacterial species, as with most models, there are some significant variations. Perhaps the best example of this comes from an analysis of the SOS-like system of the developmental organism, Bacillus subtilis. Accordingly, the most striking difference is the complex developmental regulation of the SOS system as this organism differentiates into its competent state. In this review we have given an overview of the elements that comprise the SOS system of B. subtilis. Additionally, we have summarized our most recent findings regarding the regulation of this regulon. Using these results along with new findings from other laboratories we have provided provocative molecular models for the regulation of the B. subtilis SOS system in response to DNA damage and during competent cell formation.     

Exchange of individual ribosomal proteins between ribosomes as studied by heavy isotope-transfer experiments

Summary We have described previously an inducible response in Escherichia coli which occurs during growth on low levels of the methylating agent, N-methyl-N-nitro-N-nitrosoguanidine (MNNG), and which enables cells both to survive better and to be less mutated by a subsequent challenge dose of MNNG than control cultures (Samson and Cairns, 1977). We show here that this response is distinct from previously characterised pathways of DNA repair, and particularly from the SOS response, which is another inducible effect resulting from DNA damage. An examination of the cross-reactivity of this response with other mutagens has shown that it is a generalised mechanism affecting alkylation damage to DNA. It cannot, however, be induced by UV or the UV-mimetic mutagen, 4-nitroquinoline 1-oxide, nor act on lesions put into DNA by those mutagens.     

Role of constitutive and inducible repair in radiation resistance of Escherichia coli

Radiation resistance of Escherichia coil cells depends on how efficiently DNA is recovered after damage, which is determined by the function of constitutive and inducible repair branches. The effects of additional mutations of the key genes of constitutive and inducible repair (recA, lexA, recB, polA, lig, gyr, recE, recO, recR, recJ, recQ, uvrD, helD, recN, and ruv) on radiation resistance were studied in E. coli K-12 strain AB 1157 and highly radiation-resistant isogenic strain Gam(r)444. An optimal balance ensuring a high gamma resistance of the Gam(r)444 radiation-resistant E. coli mutant was due to expression of the key SOS repair genes (recA, lexA, recN, and ruv) and activation of the presynaptic functions of the RecF homologous recombination pathway as a result of a possible mutation of the uvrD gene, which codes for repair helicase II.     

SOS independent survival against mitomycin C induced lethality in a rifampicin-nalidixic acid-resistant mutant of Escherichia coli

A combination of specific rifampicin-resistant (rpoB87) and nalidixic acid-resistant (gyrA87) mutations results in a marked increase in the survival of Escherichia coli against mitomycin C-induced lethality in mutants defective for SOS induction and excision repair. Although the response does not seem to be obligatorily dependent upon the RecA protein, the efficiency is markedly increased in its presence, even in a conventionally inactive form. This response is not elicited against lethality due to ultraviolet radiation or N-methyl-N' -nitro-N-nitrosoguanidine exposure. The combination of rpoB87 and gyrA87 mutations also greatly alleviates post-mitomycin C degradation of DNA under SOS non-inducible conditions. It is proposed that the rpoB subunit of RNA polymerase and gyrA subunit of DNA gyrase could participate in the repair of certain types of DNA damage, such as cross-links, in a mode independent of SOS-regulated excision repair and post-replication repair.     

Repair promoted by plasmid pKM101 is different from SOS repair.

In E. coli K12 bacteria carrying plasmid pKM101, prophage lambda was induced at UV doses higher than in plasmid-less parental bacteria. UV-induced reactivation per se was less effective. Bacteria with pKM101 showed no alteration in their division cycle. Plasmid pKM101 coded for a constitutive error-prone repair different from the inducible error-prone repair called SOS repair. Plasmid pKM101 protected E. coli bacteria from UV damage but slightly sensitized them to X-ray lesions. Protection against UV damage was effective in mutant bacteria deficient in DNA excision-repair provided that the recA, lexA and uvrE genes were functional. Survival of phages lambda and S13 after UV irradiation was enhanced in bacteria carrying plasmid pKM101; phage lambda mutagenesis was also increased. Plasmid pKM101 repaired potentially lethal DNA lesions, although wild-type DNA sequences may not necessarily be restored; hence the mutations observed are the traces of the original DNA lesions.     

The SOS system

In the bacterium Escherichia coli DNA damaging treatments such as ultraviolet or ionizing radiation induce a set of functions called collectively the SOS response, reviewed here. The regulation of the SOS response involves a repressor, the LexA protein, and an inducer, the RecA protein. After DNA damage an effector molecule is produced--possibly single stranded DNA--which activates the RecA protein to a form capable of catalysing proteolytic cleavage of LexA. The repressors of certain temperate prophages are cleaved under the same conditions, resulting in lysogenic induction. SOS functions are involved in DNA repair and mutagenesis, in cell division inhibition, in recovery of normal physiological conditions after the DNA damage is repaired, and possibly in cell death when DNA damage is too extensive. The SOS response also includes several chromosomal genes of unknown function, a number of plasmid encoded genes (bacteriocins, mutagenesis), and lysogenic induction of certain prophages. DNA damaging treatments seem to induce DNA repair and mutagenic activities and proviral development in many species, including mammalian cells. In general, substances which are genotoxic to higher eukaryotes induce the SOS response in bacteria. This correlation is the basis of the numerous bacterial tests for genotoxicity and carcinogenicity.     

Toxicity and mutagenicity of plumbagin and the induction of a possible new DNA repair pathway in Escherichia coli.

Actively growing Escherichia coli cells exposed to plumbagin, a redox cycling quinone that increases the flux of O2- radicals in the cell, were mutagenized or killed by this treatment. The toxicity of plumbagin was not found to be mediated by membrane damage. Cells pretreated with plumbagin could partially reactivate lambda phage damaged by exposure to riboflavin plus light, a treatment that produces active oxygen species. The result suggested the induction of a DNA repair response. Lambda phage damaged by H2O2 treatment were not reactivated in plumbagin-pretreated cells, nor did H2O2-pretreated cells reactivate lambda damaged by treatment with riboflavin plus light. Plumbagin treatment did not induce lambda phage in a lysogen, nor did it cause an increase in beta-galactosidase production in a dinD::Mu d(lac Ap) promoter fusion strain. Cells pretreated with nonlethal doses of plumbagin showed enhanced survival upon exposure to high concentrations of plumbagin, but were unchanged in their susceptibility to far-UV irradiation. polA and recA mutants were not significantly more sensitive than wild type to killing by plumbagin. However, xth-1 mutants were partially resistant to plumbagin toxicity. It is proposed that E. coli has an inducible DNA repair response specific for the type of oxidative damage generated during incubation with plumbagin. Furthermore, this response appears to be qualitatively distinct from the SOS response and the repair response induced by H2O2.