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.     

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.     

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.     

DNA loop repair by Escherichia coli cell extracts

The nick-directed DNA repair efficiency of a set of M13mp18-derived heteroduplexes containing 8-, 12-, 16-, 22-, 27-, 45-, and 429-nucleotide loops was determined by in vitro assay. Unpaired nucleotides of each heteroduplex reside within overlapping recognition sites for two restriction endonucleases, permitting independent evaluation of repair occurring on either DNA strand. Our results show that a strand break located either 3' or 5' to the loop is sufficient to direct heterology repair to the nicked strand in Escherichia coli extracts. Strand-specific repair by this system requires Mg2+ and the four dNTPs and is equally efficient on insertions and deletions. This activity is distinct from the MutHLS mismatch repair pathway. Strand specificity and repair efficiency are largely independent of the GATC methylation state of the DNA and presence of the products of mismatch repair genes mutH, mutL, and mutS. This study provides evidence for a loop repair pathway in E. coli that is distinct from conventional mismatch repair.     

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.     

Methyl-directed DNA mismatch repair in Escherichia coli

Some of the molecular aspects of methyl-directed mismatch repair in E. coli have been characterized. These include: mismatch recognition by mutS protein in which different mispairs are bound with different affinities; the direct involvement of d(GATC) sites; and strand scission by mutH protein at d(GATC) sequences with strand selection based on methylation of the DNA at those sites. In addition, communication over a distance between a mismatch and d(GATC) sites has been implicated. Analysis of mismatch correction in a defined system (Lahue et al., unpublished) should provide a direct means to further molecular aspects of this process.     

Induction and repair of double- and single-strand DNA breaks in bacteriophage lambda superinfecting Escherichia coli

Induction and repair of double- and single-strand DNA breaks have been measured after decays of 125I and 3H incorporated into the DNA and after external irradiation with 4 MeV electrons. For the decay experiments, cells of wild type Escherichia coli K-12 were superinfected with bacteriophage lambda DNA labelled with 5'-(125I)iodo-2'-deoxyuridine or with (methyl-3H)thymidine and frozen in liquid nitrogen. Aliquots were thawed at intervals and lysed at neutral pH, and the phage DNA was assayed for double- and single-strand breakage by neutral sucrose gradient centrifugation. The gradients used allowed measurements of both kinds of breaks in the same gradient. Decays of 125I induced 0.39 single-strand breaks per double-strand break. No repair of either break type could be detected. Each 3H disintegration caused 0.20 single-strand breaks and very few double-strand breaks. The single-strand breaks were rapidly rejoined after the cells were thawed. For irradiation with 4 MeV electrons, cells of wild type E. coli K-12 were superinfected with phage lambda and suspended in growth medium. Irradiation induced 42 single-strand breaks per double-strand break. The rates of break induction were 6.75 x 10(-14) (double-strand breaks) and 2.82 x 10(-12) (single-strand breaks) per rad and per dalton. The single-strand breaks were rapidly repaired upon incubation whereas the double-strand breaks seemed to remain unrepaired. It is concluded that double-strand breaks in superinfecting bacteriophage lambda DNA are repaired to a very small extent, if at all.     

Carcinogen-induced DNA repair in nucleotide-permeable Escherichia coli cells. Induction of DNA repair by the carcinogens methyl and ethyl nitrosourea and methyl methanesulfonate.

Ether-permeabilized (nucleotide-permeable) cells of Escherichia coli show excision repair of their DNA after having been exposed to the carcinogens N-methyl-N-nitrosourea (MeNOUr), N-ethyl-N-nitrosourea (EtNOUr) and methyl methanesulfonate (MeSO2OMe) which are known to bind covalently to DNA. Defect mutations in genes uvrA, uvrB, uvrC, recA, recB, recC and rep did not inhibit this excision repair. Enzymic activities involved in this repair were identified by measuring size reduction of DNA, DNA degradation to acid-soluble nucleotides and repair polymerization. 1. In permeabilized cells methyl and ethyl nitrosourea induced endonucleolytic cleavage of endogenous DNA, as determined by size reduction of denatured DNA in neutral and alkaline sucrose gradients. An enzymic activity from E. coli K-12 cell extracts was purified (greater than 2000-fold) and was found to cleave preferentially methyl-nitrosourea-treated DNA and to convert the methylated supercoiled DNA duplex (RFI) of phage phiX 174 into the nicked circular form. 2. Degradation of alkylated cellular DNA to acid solubility was diminished in a mutant lacking the 5' leads to 3' exonucleolytic activity of DNA polymerase I but was not affected in a mutant which lacked the DNA polymerizing but retained the 5' leads 3' exonucleolytic activity of DNA polymerase I. 3. An easily measurable effect is carcinogen-induced repair polymerization, making it suitable for detection of covalent binding of carcinogens and potentially carcinogenic compounds.     

Study of the effect of mutations in DNA repair genes on the thermal induction of error-free repair inEscherichia coli

Previous studies from this laboratory have shown that heat shock can induce alon gene-dependent, error-free DNA repair process in certain strains ofEscherichia coli. Further experiments have now shown that the phenomenon is dependent upon therecA, uvrA, anduvrB genes. However,lexA^– anduvrC^– strains still show the effect, although at a reduced level, so the corresponding proteins may not be essential. None of the mutations affects the induction of thermotolerance; this proves that two separate pathways are involved.     

Inducible repair of O-alkylated DNA pyrimidines in Escherichia coli

The three miscoding alkylated pyrimidines O2-methylcytosine, O2-methylthymine and O4-methylthymine are specifically recognized by Escherichia coli DNA repair enzymes. The activities are induced as part of the adaptive response to alkylating agents. O2-Methylcytosine and O2-methylthymine are removed by a DNA glycosylase, the alkA+ gene product, which also acts on N3-methylated purines. O4-Methylthymine is repaired by a methyltransferase, previously known to correct O6-methylguanine by transfer of the methyl group to one of its own cysteine residues. It is proposed that certain common structural features of the various methylated bases allow each of the two inducible repair enzymes to recognize and remove several different kinds of lesions from alkylated DNA.     

p21 promotes error-free replication-coupled DNA double-strand break repair

p21 is a well-established regulator of cell cycle progression. The role of p21 in DNA repair, however, remains poorly characterized. Here, we describe a critical role of p21 in a replication-coupled DNA double-strand break (DSB) repair that is mechanistically distinct from its cell cycle checkpoint function. We demonstrate that p21-deficient cells exhibit elevated chromatid-type aberrations, including gaps and breaks, dicentrics and radial formations, following exposure to several DSB-inducing agents. p21(-/-) cells also exhibit an increased DNA damage-inducible DNA-PK(CS) S2056 phosphorylation, indicative of elevated non-homologous DNA end joining. Concomitantly, p21(-/-) cells are defective in replication-coupled homologous recombination (HR), exhibiting decreased sister chromatid exchanges and HR-dependent repair as determined using a crosslinked GFP reporter assay. Importantly, we establish that the DSB hypersensitivity of p21(-/-) cells is associated with increased cyclin-dependent kinase (CDK)-dependent BRCA2 S3291 phosphorylation and MRE11 nuclear foci formation and can be rescued by inhibition of CDK or MRE11 nuclease activity. Collectively, our results uncover a novel mechanism by which p21 regulates the fidelity of replication-coupled DSB repair and the maintenance of chromosome stability distinct from its role in the G1-S phase checkpoint.