Induction of error-free DNA repair inEscherichia coli by Nonmutagenic Stress

Our previous studies have shown that heat shock and nutritional stress produce an increase in UV resistance and a decrease in UV-induced mutation frequency in DNA repairproficient strains ofEscherichia coli K12. The effect depends on nucleotide excision repair and requires protein synthesis. We now show that comparable changes occur after oxidation stress, exposure to ethanol, or osmotic shock, all in conditions that do not affect the natural mutation frequency. The results support the hypothesis that many unrelated, nonmutagenic treatments elicit a common protective response in these cells that involves induction of an error-free DNA excision repair system.     

Induction of error-free DNA repair inEscherichia coli by heat shock or nutritional stress

Sterilization values were determined forLegionella pneumophila in chlorine-free, chlorine-demand-free water at elevated temperatures. These values were calculated from experimentally determined D values of 2500 min, 380 min, 13.93 min, 0.74 min, and 0.45 min at 45°C, 50°C, 55°C, 60°C, and 66°C, respectively. D values, Z value and temperature coefficient do not indicate unusual thermal resistance. Sterilization values, the minimum time required to eliminate an aquatic population ofL. pneumophila at a given test temperature, indicate that temperatures greater than about 65°C may not be necessary for efficient disinfection of potable quality water. These values and monitoring of time and temperature parameters can help predict the efficacy of in situ heat treatment of potable quality waters harboringL. pneumophila.     

Induction of error-free DNA repair in Escherichia coli by thiamine deprivation

Incubation of Escherichia coli AB1157 in a thiamine-deficient medium causes a large, time-dependent increase in resistance to UV-radiation (254 nm) and a fall in its UV-induced mutation frequency to histidine prototrophy which are abolished in its uvrA mutant, but only delayed in lon- and recA- cells. The response of the lexA3 mutant resembles that of the parental cells. These effects are very similar to those we have shown to be induced by heat shock and are clearly due to an error-free, DNA-excision repair-dependent process. They may represent a general response to non-mutagenic stress in these cells.     

Starvation as an inducer of error-free DNA repair in Escherichia coli

Previous work in this laboratory has shown that heat shock or vitamin B1 deprivation induces an error-free DNA-repair process in Escherichia coli. The system is absolutely dependent on excision repair, while its induction is delayed in lon- or recA- cells. We have now shown that starvation of E. coli for amino acids, glucose or phosphate, conditions known to induce the stringent response or the glu and pho regulons, respectively, leads to a similar uvrA-dependent increase in UV resistance and decrease in UV-induced mutation frequency. These results support the hypothesis that the effect is a general response to non-mutagenic stress that may play an important role in the survival of cells exposed to harsh environments.     

Dependence on thelon gene of the thermal induction of resistance to ultraviolet light inEscherichia coli

Our previous studies have shown thatEscherichia coli JE1011 possesses an errorfree DNA repair system that is inducible by heat shock or thiamine deprivation. However, it appears to be lacking inE. coli B, which islon ^–. We now show that a similar, thermally inducible, error-free system is present inE. coli AB1157, although it requires more severe heat shock for its induction. Thelon mutant of this strain is similar toE. coli B and does not become more UV-resistant after heat shock, so this gene appears to play an essential role in the process. All three strains become more resistant to heat inactivation at 55°C following a 30°C48°C heat shock; this confirms that the induced UV and thermal resistances must arise by different mechanisms.     

Mutagenic and error-free DNA repair in Streptomyces

Summary Two mutants of Streptomyces fradiae defective in DNA repair have been characterized for their responses to the mutagenic and lethal effects of several chemical mutagens and ultraviolet (UV) light. S. fradiae JS2 (mcr-2) was more sensitive than wild type to agents which produce bulky lesions resulting in large distortions of the double helix [i.e. UV-light, 4-nitroquinoline-1-oxide (NQO), and mitomycin C (MC)] but not to agents which produce small lesions [i.e. hydroxylamine (HA), methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS) and N-methyl-N-nitro-N-nitrosoguanidine (MNNG)]. JS2 expressed a much higher frequency of mutagenesis induced by UV-light at low doses and thus appeared to be defective in an error-free excision repair pathway for bulky lesions analogous to the uvr ABC pathway of Escherichia coli. S. fradiae JS4 (mcr-4) was defective in repair of damage by most agents which produce small or bulky lesions (i.e., HA, NQO, MMS, MNNG, MC, and UV, but not EMS). JS4 was slightly hypermutable by EMS and MMS but showed reduced mutagenesis by NQO and HA. This unusual phenotype suggests that the mcr-4 ⁺ protein plays some role in error-prone repair in S. fradiae.     

Expression of theunc genes inEscherichia coli

Theunc (or atp) operon ofEscherichia coli comprises eight genes encoding the known subunits of the proton-translocating ATP synthase (H⁺-ATPase) plus a ninth gene (uncI) of unknown function. The subunit stoichiometry of the H⁺-ATPase ( ₃₃₁₁₁a₁b₂c_10–15) requires that the respectiveunc genes be expressed at different rates. This review discusses the experimental methods applied to determining how differential synthesis is achieved, and evaluates the results obtained. It has been found that the primary level of control is translational initiation. The translational efficiencies of theunc genes are determined by primary and secondary mRNA structures within their respective translational initiation regions. The respective rates of translation are matched to the subunit requirements of H⁺-ATPase assembly. Finally, points of uncertainty remain and experimental strategies which will be important in future work are discussed.     

Dipyrone-induced changes in DNA repair and other cell membrane associated processes inEscherichia coli

The analgesic, dipyrone (l,phenyl-2,3-dimethyl-5-pyrazolone-4-methylamino methane sulphonate sodium), at 20 mM concentration, inhibited the rejoining of single-strand scissions in DNA ofEscherichia coli B/r cells induced by 20 krad gamma-radiation. The chemical altered the cell membrane structure as evidenced from the uptake of acriflavin, the efflux of potassium ions from the bacterial cells and the inhibition of alkaline phosphatase-a cell membrane associated enzyme.     

Study of the DNA primary structure effect on induction of mutations by alkylating agents

Analysis and comparison of mutation spectra is one of the major tasks of molecular biology, since mutation spectra often reveal important properties of various mutagens and proteins involved in the repair/replication systems. Mutability is known to vary significantly along the nucleotide sequence. Mutations are abundant at certain positions (mutation hotspots). In this work, we applied regression analysis based on the basic logic patterns to understand the role of the nucleotide sequence context in mutation induction. The spectra of mutations induced by various alkylating agents were studied. The nucleotide bases at positions -2, -1, +1 and +2 were shown to have the most significant effect in G:C-->A:T replacements.     

Simultaneous induction of three catabolic enzymes inEscherichia coli

During a simultaneous induction of three enzymes which are subject to catabolite repression (β-galactosidase, tryptophanase and amylomaltase, or β-galactosidase, tryptophanase and D-serine deaminase) in a batch culture, the rates of synthesis of β-galactosidase and tryptophanase decreases, while the rates of synthesis of amylomaltase and D-serine deaminase remain unaffected. The addition of cAMP brings about a considerable increase of the rate of synthesis of D-serine deaminase and a partial synthesis rate increase of β-galactosidase while the synthesis rate of tryptophanase remains lowered and the synthesis rate of amylomaltase remains unaffected. In a continuous culture β-galactosidase, tryptophanase andD-serine deaminase are synthesized simultaneously at a maximum rate without mutual influence. The addition of cAMP increases the rate of synthesis of all three enzymes.     

Hydrogen peroxide induces the repair of UV-damaged DNA inEscherichia coli: AlexA-independent butuvrA- andrecA-dependent mechanism

Pretreatment with 2.5mm H₂O₂ protects bacterial cells against UV killing, a phenomenon that is independent of the SOS response. This protection possibly involves the induction of some other DNA repair mechanism, sincelexA (Ind^–) mutants pretreated with this concentration of H₂O₂ enhance the repair of UV-damaged phages. Moreover, the induction of this DNA repair mechanism is independent of theoxyR regulon. However, the repair of UV-damaged phages is not enhanced inrecA anduvrA mutants, suggesting a DNA repair mechanism independent of LexA cleavage or OxyR activation, but dependent on RecA and UvrA proteins.     

Mutagenic DNA repair in escherichia coli. V. Mutation frequency decline and error-free post-replication repair in an excision-proficient strain.

Mutation frequency decline (MFD) is an irreversible loss of newly-induced suppressor mutations occurring in excision-proficient Escherichia coli during a short period of incubation in minimal medium before plating on broth- or Casamino acids-enriched selective agar. It is known that MFD of UV-induced mutations may occur before DNA containing pre-mutagenic lesions is replicated, but we conclude that MFD can also occur after the damaged DNA has been replicated on the basis of the following evidence. (1) Mutation fixation in rich medium (i.e., loss of susceptibility to mutation frequency decline) with ethyl methanesulphonate mutagenesis begins immediately, whereas with UV it is delayed for 20--30 min. (2) The delay in mutation fixation after UV can be explained neither by inhibition of DNA replication nor by a delay in the appearance of error-prone repair activity in the irradiated population. (3) MFD at later times after UV irradiation is more rapid and is less strongly inhibited by caffeine than is MFD immediately after irradiation. (4) Excision is virtually complete 20 min after 3 J m-2 UV but at that time virtually all mutations are still susceptible to MFD. We have presented evidence elsewhere that in bacteria there is an alternative error-free excision-dependent type of post-replication repair of potentially mutagenic daughter strand gaps. We suggest that this process is inhibited at tRNA loci in the presence of nutrient broth or Casamino acids, possibly because of a broth-dependent change in the structure of the single-stranded region including the tRNA locus.     

Intracellular location of NRl-plasmid DNA inEscherichia coli minicells

Intracellular location of plasmid NR1 (M = 58 Mg/mol, stringent control of replication, 1–2 copies perEscherichia coli chromosomal equivalent) was studied and compared with that of plasmid R6KΔ1 (M = 21 Mg/mol, relaxed control of replication, 10–15 copies perE. coli chromosomal equivalent), both inE. coli minicells. Considerable difference in relative distribution of molecules of these two plasmid DNA’s between the cytoplasm and the membrane fraction was found when components of the corresponding minicell lyzates were fractionated by sedimentation in a double-linear gradient of caesium chlorid and sucrose. Also the difference in relative numbers of NR1 DNA and R6KΔ1 DNA molecules stably associated with the membrane of minicells, determined by electron-microscopic examination of the fractions containing plasmid DNA-membrane complexes, was evaluated as statistically significant. The association of NR1 DNA molecules withE. coli minicell membrane was found to be a much more frequent event than such association of R6KΔ1 molecules. The absolute amount of plasmid DNA associated with membrane in a single minicell corresponds to one molecule for both NR1 and R6KAΔ1.     

Escherichia coli DNA topoisomerase I mutants have compensatory mutations in DNA gyrase genes

Escherichia coli deletion mutants lacking DNA topoisomerase I have been identified previously and shown to grow at a normal rate. We show that such strains grow normally only because of spontaneously arising mutations that compensate for the topoisomerase I defect. Several of these compensatory mutations have been found to map at or near the genes encoding DNA gyrase, gyrA and gyrB. DNA gyrase assays of crude extracts show that strains carrying the mutations have lower gyrase activity. Thus the mutations are in the gyrase structural genes or in nearby regulatory sequences. These results, in conjunction with DNA supercoiling measurements of others, indicate that in vivo DNA superhelicity is a result of a balance between topoisomerase I and gyrase activities. An excess of negative supercoils due to an absence of topoisomerase I is deleterious to the cell, but a moderate gyrase deficiency is not harmful.     

The effect of bacteriophage induction on the repair ability ofEscherichia coli 15 TAU

The growth, lysis and survival ofEscherichia coli 15 TAU following UV irradiation was examined. An attempt was made to separate the repair processes and prophage induction by preincubation with chloramphenicol and subsequent incubation with chloramphenicol or caffeine. It was found that the prophage induction does not diminish the repair ability of cells that have survived the induction. It cannot be ascertained whether the cell fraction was induced at all or whether the induction was partially reverted subsequently. It can be concluded that the results derived from examining the survival of tially reverted subsequently. It can be concluded that the results derived from examining the survival of UV-irradiation cultures ofEscherichia coli 15 TAI do not require any correction for phage induction.     

Mismatch repair participates in error-free processing of DNA interstrand crosslinks in human cells

DNA interstrand crosslinks (ICLs) present formidable blocks to DNA metabolic processes and must be repaired for cell survival. ICLs are induced in DNA by intercalating compounds such as the widely used therapeutic agent psoralen. In bacteria, both nucleotide excision repair (NER) and homologous recombination are required for the repair of ICLs. The processing of ICLs in mammalian cells is not clearly understood. However, it is known that processing can occur by NER, which for psoralen ICLs can be an error-generating process conducive to mutagenesis. We show here that another repair pathway, mismatch repair (MMR), is also involved in eliminating psoralen ICLs in human cells. MMR deficiency renders cells hypersensitive to psoralen ICLs without diminishing their mutagenic potential, suggesting that MMR does not contribute to error-generating repair, and that MMR may represent a relatively error-free mechanism for processing these lesions in human cells. Thus, enhancement of MMR relative to NER may reduce the mutagenesis caused by DNA ICLs in humans.