scholarly journals The multiple activities of Escherichia coli endonuclease IV and the extreme lability of 5′-terminal base-free deoxyribose 5-phosphates

1989 ◽  
Vol 259 (3) ◽  
pp. 761-768 ◽  
Author(s):  
V Bailly ◽  
W G Verly
Keyword(s):  
Escherichia Coli ◽  
Dna Polymerase ◽  
Rat Liver ◽  
Half Life ◽  
Elimination Reaction ◽  
Ap Endonuclease ◽  
Exonuclease Iii ◽  
E Coli ◽  
Beta Elimination ◽  
Ap Site

Escherichia coli endonuclease IV hydrolyses the C(3′)-O-P bond 5′ to a 3′-terminal base-free deoxyribose. It also hydrolyses the C(3′)-O-P bond 5′ to a 3′-terminal base-free 2′,3′-unsaturated sugar produced by nicking 3′ to an AP (apurinic or apyrimidinic) site by beta-elimination; this explains why the unproductive end produced by beta-elimination is converted by the enzyme into a 3′-OH end able to prime DNA synthesis. The action of E. coli endonuclease IV on an internal AP site is more complex: in a first step the C(3′)-O-P bond 5′ to the AP site is hydrolysed, but in a second step the 5′-terminal base-free deoxyribose 5′-phosphate is lost. This loss is due to a spontaneous beta-elimination reaction in which the enzyme plays no role. The extreme lability of the C(3′)-O-P bond 3′ to a 5′-terminal AP site contrasts with the relative stability of the same bond 3′ to an internal AP site; in the absence of beta-elimination catalysts, at 37 degrees C the half-life of the former is about 2 h and that of the latter 200 h. The extreme lability of a 5′-terminal AP site means that, after nicking 5′ to an AP site with an AP endonuclease, in principle no 5′----3′ exonuclease is needed to excise the AP site: it falls off spontaneously. We have repaired DNA containing AP sites with an AP endonuclease (E. coli endonuclease IV or the chromatin AP endonuclease from rat liver), a DNA polymerase devoid of 5′----3′ exonuclease activity (Klenow polymerase or rat liver DNA polymerase beta) and a DNA ligase. Catalysts of beta-elimination, such as spermine, can drastically shorten the already brief half-life of a 5′-terminal AP site; it is what very probably happens in the chromatin of eukaryotic cells. E. coli endonuclease IV also probably participates in the repair of strand breaks produced by ionizing radiations: as E. coli endonuclease VI/exonuclease III, it is a 3′-phosphoglycollatase and also a 3′-phosphatase. The 3′-phosphatase activity of E. coli endonuclease VI/exonuclease III and E. coli endonuclease IV can also be useful when the AP site has been excised by a beta delta-elimination reaction.

scholarly journals Escherichia coli endonuclease III is not an endonuclease but a β-elimination catalyst

1987 ◽  
Vol 242 (2) ◽  
pp. 565-572 ◽  
Author(s):  
V Bailly ◽  
W G Verly
Keyword(s):  
Dna Polymerase ◽  
Exonuclease Activity ◽  
Alkaline Ph ◽  
Sugar Phosphate ◽  
Exonuclease Iii ◽  
E Coli ◽  
Endonuclease Iii ◽  
Ap Sites ◽  
Beta Elimination ◽  
Ap Site

The oligonucleotide [5′-32P]pdT8d(-)dTn, containing an apurinic/apyrimidinic (AP) site [d(-)], yields three radioactive products when incubated at alkaline pH: two of them, forming a doublet approximately at the level of pdT8dA when analysed by polyacrylamide-gel electrophoresis, are the result of the beta-elimination reaction, whereas the third is pdT8p resulting from beta delta-elimination. The incubation of [5′-32P]pdT8d(-)dTn, hybridized with poly(dA), with E. coli endonuclease III yields two radioactive products which have the same electrophoretic behaviour as the doublet obtained by alkaline beta-elimination. The oligonucleotide pdT8d(-) is degraded by the 3′-5′ exonuclease activity of T4 DNA polymerase as well as pdT8dA, showing that a base-free deoxyribose at the 3′ end is not an obstacle for this activity. The radioactive products from [5′-32P]pdT8d(-)dTn cleaved by alkaline beta-elimination or by E. coli endonuclease III are not degraded by the 3‘-5’ exonuclease activity of T4 DNA polymerase. When DNA containing AP sites labelled with 32P 5′ to the base-free deoxyribose labelled with 3H in the 1′ and 2′ positions is degraded by E. coli endonuclease VI (exonuclease III) and snake venom phosphodiesterase, the two radionuclides are found exclusively in deoxyribose 5-phosphate and the 3H/32P ratio in this sugar phosphate is the same as in the substrate DNA. When DNA containing these doubly-labelled AP sites is degraded by alkaline treatment or with Lys-Trp-Lys, followed by E. coli endonuclease VI (exonuclease III), some 3H is found in a volatile compound (probably 3H2O) whereas the 3H/32P ratio is decreased in the resulting sugar phosphate which has a chromatographic behaviour different from that of deoxyribose 5-phosphate. Treatment of the DNA containing doubly-labelled AP sites with E. coli endonuclease III, then with E. coli endonuclease VI (exonuclease III), also results in the loss of 3H and the formation of a sugar phosphate with a lower 3H/32P ratio that behaves chromatographically as the beta-elimination product digested with E. coli endonuclease VI (exonuclease III). From these data, we conclude that E. coli endonuclease III cleaves the phosphodiester bond 3′ to the AP site, but that the cleavage is not a hydrolysis leaving a base-free deoxyribose at the 3′ end as it has been so far assumed. The cleavage might be the result of a beta-elimination analogous to the one produced by an alkaline pH or Lys-Trp-Lys. Thus it would seem that E. coli ‘endonuclease III’ is, after all, not an endonuclease.

scholarly journals The use of thioglycollate to demonstrate DNA AP (apurinic/apyrimidinic-site) lyase activities. Biological consequences of thiol addition to the 5′ product of a β-elimination reaction at an AP site in DNA

1991 ◽  
Vol 273 (3) ◽  
pp. 777-782 ◽  
Author(s):  
S Bricteux-Grégoire ◽  
W G Verly
Keyword(s):  
Escherichia Coli ◽  
Elimination Reaction ◽  
Ap Endonuclease ◽  
Lyase Activity ◽  
Ap Sites ◽  
Ap Lyase ◽  
Ap Site

Thioglycollate reacts with the 5′ product of AP lyase activity on apurinic/apyrimidinic (AP) sites in DNA. The 3′-terminal thioglycollate-unsaturated sugar 5-phosphate adduct can be released by the use of Escherichia coli endonuclease IV or endonuclease VI, and identified by DEAE-Sephadex chromatography. In contrast, the mammalian AP endonuclease is unable to excise a 3′-terminal thiol-unsaturated sugar adduct; this lesion, which must sometimes occur in vivo, might be irreparable and have pathological consequences.

scholarly journals OmpC regulation differs between ST131 and non-ST131 Escherichia coli clinical isolates and involves differential expression of the small RNA MicC

2020 ◽  
Vol 75 (5) ◽  
pp. 1151-1158
Author(s):  
Corey S Suelter ◽  
Nancy D Hanson
Keyword(s):  
Escherichia Coli ◽  
Half Life ◽  
Selective Advantage ◽  
Resistance Mechanisms ◽  
Northern Blotting ◽  
Rt Pcr ◽  
E Coli ◽  
Protein Levels ◽  
Porin Proteins ◽  
Mrna Half Life

Abstract Background Virulence genes and the expression of resistance mechanisms undoubtedly play a role in the successful spread of the pandemic clone Escherichia coli ST131. Porin down-regulation is a chromosomal mechanism associated with antibiotic resistance. Translation of porin proteins can be impacted by modifications in mRNA half-life and the interaction among small RNAs (sRNAs), the porin transcript and the sRNA chaperone Hfq. Modifications in the translatability of porin proteins could impact the fitness and therefore the success of E. coli ST131 isolates in the presence of antibiotic. Objectives To identify differences in the translatability of OmpC and OmpF porins for different STs of E. coli by comparing steady-state RNA levels, mRNA half-life, regulatory sRNA expression and protein production. Methods RNA expression was evaluated using real-time RT–PCR and OmpC mRNA half-life by northern blotting. OmpC, OmpF and Hfq protein levels were evaluated by immunoblotting. Results Differences between ST131 and non-ST131 isolates included: (i) the level of OmpC RNA and protein produced with mRNA expression higher for ST131 but OmpC protein levels lower compared with non-ST131 isolates; (ii) OmpC mRNA half-life (21–30 min for ST131 isolates compared with <2–23 min for non-ST131 isolates); and (iii) levels of the sRNA MicC (2- to 120-fold for ST131 isolates compared with −4- to 70-fold for non-ST131 isolates). Conclusions Mechanisms involved in the translatability of porin proteins differed among different STs of E. coli. These differences could provide a selective advantage to ST131 E. coli when confronted with an antibiotic-rich environment.

scholarly journals Effects of polyamines and methylglyoxal bis(guanylhydrazone) on hepatic nuclear structure and deoxyribonucleic acid template activity

1975 ◽  
Vol 151 (3) ◽  
pp. 505-512 ◽  
Author(s):  
K B Brown ◽  
N F Nelson ◽  
D G Brown
Keyword(s):  
Dna Polymerase ◽  
Rat Liver ◽  
Template Activity ◽  
Isolated Rat Liver ◽  
E Coli ◽  
Rat Liver Nuclei ◽  
Calf Thymus ◽  
Liver Nuclei ◽  
Dna Template ◽  
Isolated Rat

1. The interaction of polyamines and methylglyoxal bis(guanythydrazone) (1, 1′-[(methylethanediylidene)-dinitrilo]diguanidine) with isolated rat liver nuclei was investigated by electron microscopy. 2. At 4mM, putrescine was without effect; however, spermidine, spermine or methylglyoxal bis(guanythydrazone) resulted in dispersed chromatin and alterations in nucleolar structure. In addition, spermidine or methylglyoxal bis(guanylhydrazone) caused marked aggregation of interchromatin granules. 3. The DNA template property of calf thymus DNA was examined by using DNA polymerases from Escherichia coli, Micrococcus lysodeikticus and calf thymus in the presence of 0-5 mM-amine. 4. In the presence of DNA polymerase, spermine or methylglyoxal bis(guanylhydrazone) inhibited activity, whereas putrescine or spermidine had much less effect or in some cases stimulated [3H]dTMP incorporation. 5. Template activity which was inhibited by spermine or methylglyoxal bis(guanylhydrazone) could be partially restored by additional DNA or enzyme. 6. When mixed with calf thymus DNA, calf thymus histone inhibited template activity as measured with E. coli DNA polymerase. The template activity of such a ‘histone-nucleate’ could not be restored by putrescine, spermidine, spermine or methylglyoxal bis(guanylhydrazone). 7. DNA template activity of isolated rat liver nuclei was tested by using E. coli DNA polymerase. None of the amines was able to increase the template activity of the nuclear DNA in vitro.

scholarly journals Bacteriophage-T4 and Micrococcus luteus UV endonucleases are not endonucleases but β-elimination and sometimes βδ-elimination catalysts

1989 ◽  
Vol 259 (3) ◽  
pp. 751-759 ◽  
Author(s):  
V Bailly ◽  
B Sente ◽  
W G Verly
Keyword(s):  
Enzyme Preparation ◽  
Micrococcus Luteus ◽  
Bacteriophage T4 ◽  
Pyrimidine Dimer ◽  
Elimination Reaction ◽  
Ap Sites ◽  
Beta Elimination ◽  
Ap Site ◽  
Polynucleotide Chain

Bacteriophage-T4 UV endonuclease nicks the C(3′)-O-P bond 3′ to AP (apurinic or apyrimidinic) sites by a beta-elimination reaction. The breakage of this bond is sometimes followed by the nicking of the C(5′)-O-P bond 5′ to the AP site, leaving a 3′-phosphate end; delta-elimination is proposed as a mechanism to explain this second reaction. The AP site formed when this enzyme acts on a pyrimidine dimer in a polynucleotide chain undergoes the same nicking reactions. Micrococcus luteus UV endonuclease also nicks the C(3′)-O-P bond 3′ to AP sites by a beta-elimination reaction. No subsequent delta-elimination was observed, but this might be due to the presence of 2-mercaptoethanol in the enzyme preparation.

Cloning and expression of a functional rat liver D-β-hydroxybutyrate dehydrogenase-β-galactosidase fusion protein in Escherichia coli

1991 ◽  
Vol 69 (9) ◽  
pp. 670-673
Author(s):  
Sharon Churchill ◽  
Perry Churchill
Keyword(s):  
Escherichia Coli ◽  
Rat Liver ◽  
Polyclonal Antibodies ◽  
Cloning And Expression ◽  
Dependent Manner ◽  
Bacteriophage Λ ◽  
Expression Library ◽  
Expression Plasmid ◽  
E Coli ◽  
Dose Dependent

A rat liver bacteriophage λ expression library was probed using polyclonal antibodies raised to purified rat liver D-β-hydroxybutyrate dehydrogenase (BDH). A clone was selected that contained a 1.2-kb insert. The insert placed in an expression plasmid was utilized to transform Escherichia coli. These cells were shown to possess phosphatidylcholine-dependent BDH activity. Cells transformed with only the plasmid had no detectable BDH activity in the presence of phosphatidylcholine. The expressed activity in E. coli could be inhibited in a dose-dependent manner by BDH antiserum.Key words: D-β-hydroxybutyrate dehydrogenase, cloning, expression.

THE INFLUENCE OF THE PO1A1 MUTATION UPON RECOMBINATION IN ESCHERICHIA COLI K12

1979 ◽  
Vol 21 (3) ◽  
pp. 423-428 ◽  
Author(s):  
Barry W. Glickman ◽  
Tineke Rutgers
Keyword(s):  
Escherichia Coli ◽  
Dna Polymerase ◽  
Genetic Recombination ◽  
Dna Polymerase I ◽  
Multiple Gene ◽  
E Coli ◽  
Recombinational Process ◽  
K 12 ◽  
Polymerase I

Genetic recombination in Escherichia coli is a highly regulated process involving multiple gene products. We have investigated the role of DNA polymerase I in this process by studying the effect of the po1A1 mutation upon DNA transfer and conjugation in otherwise isogenic suppressor-free strains of E. coli K-12. It was found that the po1A1 mutation greatly reduces recombination in Hfr crosses (a factor of 20 in Po1+ × Po1A1 crosses and more than a factor of 100 in Po1A1 × Po1A1 crosses). However, since the po1A1 mutation reduces the strains capacity to act as a recipient for an F-prime and the analysis of recombination transfer gradients revealed no differences between Po1+ and Po1− strains, it is concluded that DNA polymerase I probably affects the transfer and/or stability of donor DNA rather than the recombinational process itself.

scholarly journals Inactivation of Umuc Protein Significantly Reduces Resistance to Ciprofloxacin and SOS Mutagenesis in Escherichia coli Mutants Harboring Intact Umud Gene

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Razieh Pourahmad Jaktaji ◽  
Sayedeh Marzieh Nourbakhsh Rezaei
Keyword(s):  
Escherichia Coli ◽  
Dna Polymerase ◽  
Genetic Background ◽  
Pcr Amplification ◽  
Sos Response ◽  
Rpob Gene ◽  
Dilution Method ◽  
E Coli ◽  
Sos Mutagenesis ◽  
Dna Polymerase V

Background: Ciprofloxacin induces SOS response and mutagenesis by activation of UmuD’2C (DNA polymerase V) and DinB (DNA polymerase IV) in Escherichia coli, leading to antibiotic resistance during therapy. Inactivation of DNA polymerase V can result in the inhibition of mutagenesis in E. coli. Objectives: The aim of this research was to investigate the effect of UmuC inactivation on resistance to ciprofloxacin and SOS mutagenesis in E. coli mutants. Methods: Ciprofloxacin-resistant mutants were produced in a umuC- genetic background in the presence of increasing concentrations of ciprofloxacin. The minimum inhibitory concentration of umuC-mutants was measured by broth dilution method. Alterations in the rifampin resistance-determing region of rpoB gene were assessed by PCR amplification and DNA sequencing. The expression of SOS genes was measured by quantitative real-time PCR assay. Results: Results showed that despite the induction of SOS response (overexpression of recA, dinB, and umuD genes) following exposure to ciprofloxacin in E. coliumuC mutants, resistance to ciprofloxacin and SOS mutagenesis significantly decreased. However, rifampicin-resistant clones emerged in this genetic background. One of these clones showed mutations in the rifampicin resistance-determining region of rpoB (cluster II). The low mutation frequency of E. coli might be associated with the presence and overexpression of umuD gene, which could somehow limit the activity of DinB, the location and type of mutations in the β subunit of RNA polymerase. Conclusions: In conclusion, for increasing the efficiency of ciprofloxacin against Gram-negative bacteria, use of an inhibitor of umuC, along with ciprofloxacin, would be helpful.

Cloning and identification of the product of the dnaE gene of Escherichia coli

1982 ◽  
Vol 152 (1) ◽  
pp. 351-356
Author(s):  
M M Welch ◽  
C S McHenry
Keyword(s):  
Escherichia Coli ◽  
Restriction Endonuclease ◽  
Dna Polymerase ◽  
Alpha Subunit ◽  
Temperature Sensitive ◽  
Restriction Map ◽  
Dna Polymerase Iii ◽  
Restriction Endonuclease Site ◽  
E Coli ◽  
A Site

We successively subcloned the dnaE gene of Escherichia coli into pBR322, resulting in a plasmid that contains 4.6 kilobases of E. coli DNA. This plasmid can complement a dnaE temperature-sensitive mutation. A restriction map of the dnaE gene and the surrounding 10.7-kilobase region of the E. coli chromosome was determined. A unique HindIII restriction endonuclease site within the cloned segment of DNA was identified as a site required for expression of the dnaE gene. By using the maxicell plasmid-directed protein synthesizing system, we demonstrated that dnaE codes for the alpha subunit of DNA polymerase III.

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