Role of mismatch repair in the Escherichia coli UVM response

Mutagenesis at 3,N4-ethenocytosine (epsilonC), a nonpairing mutagenic lesion, is significantly enhanced in Escherichia coli cells pretreated with UV, alkylating agents, or H2O2. This effect, termed UVM (for UV modulation of mutagenesis), is distinct from known DNA damage-inducible responses, such as the SOS response, the adaptive response to alkylating agents, or the oxyR-mediated response to oxidative agents. Here, we have addressed the hypothesis that UVM results from transient depletion of a mismatch repair activity that normally acts to reduce mutagenesis. To test whether the loss of mismatch repair activities results in the predicted constitutive UVM phenotype, E. coli cells defective for methyl-directed mismatch repair, for very-short-patch repair, or for the N-glycosylase activities MutY and MutM were treated with the UVM-inducing agent 1-methyl-3-nitro-1-nitrosoguanidine, with subsequent transfection of M13 viral single-stranded DNA bearing a site-specific epsilonC lesion. Survival of the M13 DNA was measured as transfection efficiency, and mutation fixation at the lesion was characterized by multiplex sequencing technology. The results showed normal UVM induction patterns in all the repair-defective strains tested. In addition, normal UVM induction was observed in cells overexpressing MutH, MutL, or MutS. All strains displayed UVM reactivation, the term used to describe the increased survival of epsilonC-containing DNA in UVM-induced cells. Taken together, these results indicate that the UVM response is independent of known mismatch repair systems in E. coli and may thus represent a previously unrecognized misrepair or misreplication pathway.     

Stationary phase induction of dnaN and recF, two genes of Escherichia coli involved in DNA replication and repair

The beta subunit of DNA polymerase III holoenzyme, the Escherichia coli chromosomal replicase, is a sliding DNA clamp responsible for tethering the polymerase to DNA and endowing it with high processivity. The gene encoding beta, dnaN, maps between dnaA and recF, which are involved in initiation of DNA replication at oriC and resumption of DNA replication at disrupted replication forks, respectively. In exponentially growing cells, dnaN and recF are expressed predominantly from the dnaA promoters. However, we have found that stationary phase induction of the dnaN promoters drastically changes the expression pattern of the dnaA operon genes. As a striking consequence, synthesis of the beta subunit and RecF protein increases when cell metabolism is slowing down. Such an induction is dependent on the stationary phase sigma factor, RpoS, although the accumulation of this factor alone is not sufficient to activate the dnaN promoters. These promoters are located in DNA regions without static bending, and the -35 hexamer element is essential for their RpoS-dependent induction. Our results suggest that stationary phase-dependent mechanisms have evolved in order to coordinate expression of dnaN and recF independently of the dnaA regulatory region. These mechanisms might be part of a developmental programme aimed at maintaining DNA integrity under stress conditions.     

Expansion and deletion of triplet repeat sequences in Escherichia coli occur on the leading strand of DNA replication

Expansions and deletions of triplet repeat sequences that cause human hereditary neurological diseases were previously suggested to be mediated by the formation of DNA hairpins on the lagging strand during replication. The replication properties of CTG.CAG, CGG.CCG, and TTC.GAA repeats were studied in Escherichia coli using an in vivo phagemid system as a model for continuous leading strand synthesis. The repeats were substantially deleted when the CTG, CGG, and GAA repeats were the templates for rolling circle replication from the f1 phage origin. The deletions may be mediated by hairpins formed by these repeat tracts. The distributions of the deletion products of the CTG.CAG and CGG.CCG tracts indicated that hairpins of discrete sizes mediate deletions during complementary strand synthesis. Deletions during rolling circle synthesis are caused by larger hairpins of specific sizes. Thus, most deletion products were of defined lengths, suggesting a preference for specific hairpin intermediates. Small expansions of the CTG.CAG and CGG.CCG repeats were also observed, presumably due to the formation of CTG and CGG hairpins on the nascent complementary strand. Since rolling circle replication has been established in vitro as a model for leading strand synthesis, we conclude that triplet repeat instability can also occur on the leading strand of DNA replication.     

急性白血病组蛋白乙酰化修饰及其对错配修复基因表达的调控作用

目的 探讨急性白血病患者组蛋白乙酰化修饰规律,并探索组蛋白乙酰化对错配修复基因hMSH2和hMLH1差异表达的调控作用.方法 用反转录-聚合酶链反应(RT-PCR)方法检测56例急性白血病患者和30名健康志愿者单个核细胞(MNC)的错配修复基因hMSH2和hMLH1 mRNA的表达,用Western blot法检测组蛋白H3、H4、去乙酰化酶(HDAC1)、hMSH2和hMLH1基因的蛋白表达情况.用组蛋白去乙酰转移酶抑制剂(TSA)诱导30例白血病患者MNC乙酰化,并检测处理后MNC的组蛋白H3、H4、HDAC1、hMSH2和hMLH1的表达状态变化.结果 急性白血病组的hMSH2和hMLH1、组蛋白H3、H4的蛋白表达量分别为0.4610±0.1211、0.4013±0.1143、0.4103±0.1241和0.4251±0.1081,均明显低于健康志愿者组的蛋白表达量(0.9461±0.1841、0.9960±0.2021、0.8971±0.1194、0.9513±0.1953),差异均有统计学意义(t值分别为3.341、3.935、2.843、3.575,P＜0.05);而急性白血病患者组的HDAC1表达(0.8841±0.2018)高于健康志愿者组的表达量(0.5142±0.1340),差异有统计学意义(t=2.634,P＜0.05);TSA作用于白血病单个核细胞后,组蛋白H3、H4、hMSH2和hMLH1的表达上调,分别比阴性对照组表达上调2.9倍、3.4倍、1.5倍和1.6倍,而HDAC1的蛋白表达出现明显的抑制,表达下调为阴性对照组的40%.结论 急性白血病患者的组蛋白乙酰化呈低表达现象,组蛋白乙酰化在急性白血病患者中对错配修复基因差异表达具有调控作用.     

The mechanism of thioredoxin reductase from human placenta is similar to the mechanisms of lipoamide dehydrogenase and glutathione reductase and is distinct from the mechanism of thioredoxin reductase from Escherichia coli

Thioredoxin reductase, lipoamide dehydrogenase, and glutathione reductase are members of the pyridine nucleotide-disulfide oxidoreductase family of dimeric flavoenzymes. The mechanisms and structures of lipoamide dehydrogenase and glutathione reductase are alike irrespective of the source (subunit M(r) approximately 55,000). Although the mechanism and structure of thioredoxin reductase from Escherichia coli are distinct (M(r) approximately 35,000), this enzyme must be placed in the same family because there are significant amino acid sequence similarities with the other two enzymes, the presence of a redox-active disulfide, and the substrate specificities. Thioredoxin reductase from higher eukaryotes on the other hand has a M(r) of approximately 55,000 [Luthman, M. & Holmgren, A. (1982) Biochemistry 21, 6628-6633; Gasdaska, P. Y., Gasdaska, J. R., Cochran, S. & Powis, G. (1995) FEBS Lett 373, 5-9; Gladyshev, V. N., Jeang, K. T. & Stadtman, T.C. (1996) Proc. Natl. Acad. Sci. USA 93, 6146-6151]. Thus, the evolution of this family is highly unusual. The mechanism of thioredoxin reductase from higher eukaryotes is not known. As reported here, thioredoxin reductase from human placenta reacts with only a single molecule of NADPH, which leads to a stable intermediate similar to that observed in titrations of lipoamide dehydrogenase or glutathione reductase. Titration of thioredoxin reductase from human placenta with dithionite takes place in two spectral phases: formation of a thiolate-flavin charge transfer complex followed by reduction of the flavin, just as with lipoamide dehydrogenase or glutathione reductase. The first phase requires more than one equivalent of dithionite. This suggests that the penultimate selenocysteine [Tamura, T. & Stadtman, T.C. (1996) Proc. Natl. Acad. Sci. USA 93, 1006-1011] is in redox communication with the active site disulfide/dithiol. Nitrosoureas of the carmustine type inhibit only the NADPH reduced form of human thioredoxin reductase. These compounds are widely used as cytostatic agents, so this enzyme should be studied as a target in cancer chemotherapy. In conclusion, three lines of evidence indicate that the mechanism of human thioredoxin reductase is like the mechanisms of lipoamide dehydrogenase and glutathione reductase and differs fundamentally from the mechanism of E. coli thioredoxin reductase.     

Codon repeats in genes associated with human diseases: fewer repeats in the genes of nonhuman primates and nucleotide substitutions concentrated at the sites of reiteration

Five human diseases are due to an excessive number of CAG repeats in the coding regions of five different genes. We have analyzed the repeat regions in four of these genes from nonhuman primates, which are not known to suffer from the diseases. These primates have CAG repeats at the same sites as in human alleles, and there is similar polymorphism of repeat number, but this number is smaller than in the human genes. In some of the genes, the segment of poly(CAG) has expanded in nonhuman primates, but the process has advanced further in the human lineage than in other primate lineages, thereby predisposing to diseases of CAG reiteration. Adjacent to stretches of homogeneous present-day codon repeats, previously existing codons of the same kind have undergone nucleotide substitutions with high frequency. Where these lead to amino acid substitutions, the effect will be to reduce the length of the original homopolymeric stretch in the protein.     

The F420H2-dehydrogenase from Methanolobus tindarius: cloning of the ffd operon and expression of the genes in Escherichia coli

Acute desensitization of contraction and its relative mechanisms have been studied in smooth muscle cells isolated from guinea pig stomach. Desensitization was induced by pre-exposure of the cells to one of the excitatory neuropeptides linked to the phospholipase C intracellular cascade, i.e., cholecystokinin (CCK), gastrin-releasing peptide, and Substance P. Desensitization was homologous after a 30-s pre-exposure and heterologous if pre-exposure lasted for 5 min or longer. Homologous desensitization was studied in a more detailed way after pre-exposure to CCK. Preincubation with increasing concentrations of CCK (10 pM-1 microM) induced a progressive rightward shift of the dose-response curves associated with both a decrease in potency (ED50 4.5 pM-2.2 nM) and a maximum response that were not related to a modification of response kinetics. After brief pre-exposure to 1 nM CCK (Dmax), an inhibition of contraction was observed in response to an identical dose of CCK (45.1 +/- 8.6%), the decreased response being associated with an inhibition of inositol phosphates and [Ca++]i mobilization. Both inositol trisphosphate (InsP3)-induced contraction and [Ca++]i mobilization were inhibited to a lesser extent than CCK-induced responses. Any longer pre-exposure of cells to one of the above-mentioned neuropeptides caused heterologous desensitization, with an observed inhibition of contraction in response to all tested agonists (CCK, 60.3 +/- 5.9%; gastrin-releasing peptide: 56.7 +/- 3. 5%; Substance P, 60.6 +/- 6.5%). A similar decrease was observed in InsP3-induced contractions resulting in a desensitization of the InsP3 response as well. Full recovery of contractile responses appeared within 30 min from the end of preincubation, thus indicating that degradation of membrane receptors did not occur. Although pre-exposure of the cells to protein kinase C inhibitor GF109203X did not modify CCK-induced homologous desensitization, it blocked CCK-induced heterologous desensitization. This study demonstrates that excitatory phospholipase C-coupled enteric neuropeptides induce a time-dependent homologous as well as heterologous desensitization of smooth muscle contraction occurring at receptor and postreceptor levels.     

Trans activation of the Escherichia coli ato structural genes by a regulatory protein from Bacillus megaterium: potential use in polyhydroxyalkanoate production

A Bacillus megaterium genomic fragment, which encoded an activator homologous to sigma 54 regulators and which was capable of activating Escherichia coli ato genes in trans, was detected in a gene library of B. megaterium screened for beta-ketothiolase activity. The fragment presented only one complete open reading frame (ORF1), which encoded a protein of 398 amino acids. The recombinant plasmid complemented mutations in the Escherichia coli atoC regulatory gene. The constitutive expression of the E. coli ato operon mediated by ORF1 could be useful for the synthesis of polyhydroxyalkanoates with different flexibility properties by recombinant E. coli strains.     

The hide-and-seek of retrospective revaluation: Recovery from blocking is context dependent in human causal learning.

In a typical blocking procedure, pairings of a compound consisting of 2 stimuli, A and X, with the outcome are preceded by pairings of only A with the outcome (i.e., A+ then AX+). This procedure is known to diminish responding to the target cue (X) relative to a control group that does not receive the preceding training with blocking cue A. We report 2 experiments that investigated the effect of extinguishing a blocking cue on responding to the target cue in a human causal learning paradigm (i.e., A+ and AX+ training followed by A– training). The results indicate that extinguishing a blocking cue increases conditioned responding to the target cue. Moreover, this increase appears to be context dependent, such that increased responding to the target is limited to the context in which extinction of the blocking cue took place. We discuss these findings in the light of associative and propositional learning theories. (PsycINFO Database Record (c) 2011 APA, all rights reserved)     

Expansion and contraction of ribosomal DNA repeats in Saccharomyces cerevisiae: requirement of replication fork blocking (Fob1) protein and the role of RNA polymerase I

Saccharomyces cerevisiae carries approximately 150 copies of rDNA in tandem repeats. It was found that the absence of an essential subunit of RNA polymerase I (Pol I) in rpa135 deletion mutants triggers a gradual decrease in rDNA repeat number to about one-half the normal level. Reintroduction of the missing RPA135 gene induced a gradual increase in repeat number back to the normal level. Gene FOB1 was shown to be essential for both the decrease and increase of rDNA repeats. FOB1 was shown previously to be required for replication fork blocking (RFB) activity at RFB site in rDNA and for recombination hot-spot (HOT1) activity. Thus, DNA replication fork blockage appears to stimulate recombination and play an essential role in rDNA expansion/contraction and sequence homogenization, and possibly, in the instability of repeated sequences in general. RNA Pol I, on the other hand, appears to control repeat numbers, perhaps by stabilizing rDNA with the normal repeat numbers as a stable nucleolar structure.     

Deletion mutations in the speED operon: spermidine is not essential for the growth of Escherichia coli

Null mutants of Escherichia coli were constructed that cannot synthesize spermidine, because of deletions in the gene encoding S-adenosylmethionine decarboxylase. These mutants are still able to grow at near normal rates in purified media deficient in polyamines. These results in E. coli differ from recent findings that null mutants of Saccharomyces cerevisiae and of Neurospora crassa have an absolute growth requirement for spermidine.     

Sulfatide from the pig jejunum brush border epithelial cell surface is involved in binding of Escherichia coli enterotoxin b

Using a quantitative dot blot overlay assay of polyvinylidene difluoride membranes, we investigated the ability of Escherichia coli heat-stable enterotoxin b (STb) to bind to various glycolipids of defined structure. STb bound strongly to acidic glycosphingolipids, including sulfatide (or 3'-sulfogalactosylceramide) and several gangliosides, but not significantly to their derivatives, galactosylceramide and asialogangliosides, respectively. STb exhibited the highest binding affinity for sulfatide. STb bound to pure sulfatide in a dose-dependent and saturable manner, with a detection level of a few nanograms. The binding was not inhibited by tetramethylurea, which is a strong disrupter of hydrophobic interactions, or by the anionic sulfated polymer of glucose, dextran sulfate, indicating that the binding is not due solely to either hydrophobic or ionic interactions via the sulfate group of the sulfatide. The specificity of the binding was confirmed by the finding that a 500-fold molar excess of sulfatide inhibited STb binding by approximately 45%, whereas no competition was obtained with galactosylceramide under the same conditions. Taken together, our data indicated that a galactose residue linked to a sulfate group is required for the binding specificity of STb. Then, total lipids extracted either from the mucous layer or from the epithelial cells of the pig jejunum brush border, the natural target of STb, were analyzed by thin-layer chromatography (TLC). Both extracts contained a lipidic molecule with a relative mobility on a TLC plate similar to that of the sulfatide standard. The migrated lipid extracted directly from a preparative TLC plate was confirmed to be sulfatide, as it was recognized by laminin, a sulfated glycolipid binding protein, and by a monoclonal antibody directed against sulfatide. In an overlay assay on PVDF membranes, STb bound to the sulfatide prepared from porcine jejunum as well as to the sulfatide standard. Thus, these findings suggest that the terminal oligosaccharide sequence Gal(3SO4)beta1- on sulfatide could mediate binding of STb to its target cells and, in support of a recent report (E. Rousset, J. Harel, and J. D. Dubreuil, Microb. Pathog. 24:277-288, 1998), probably terminal sialic acid residue on another glycosphingolipid. Moreover, pretreatment in the ligated intestinal loop assay with laminin or sulfatase altered the biological activity of STb. In summary, we present data indicating that sulfatide represents a functional receptor for the STb toxin.