Restriction and modification of a self-complementary octanucleotide containing the EcoRI substrate

Greene, Patricia J.; Poonian, Mohindar S.; Nussbaum, A. L.; Tobias, Lawrence D.; Garfin, Davi.E.; Boyer, Herbert W.; Goodman, H M

doi:10.1016/s0022-2836(75)80143-4

Cited by 161 publications

(60 citation statements)

References 22 publications

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“…7A), again indicating a preference of the EcoRI endonuclease for flanking dA residues as compared to d G residues. Oligodeoxynucleotides Ia and Ib are not cleaved at 45 "C. This result is not typical for the EcoRI-endonuclease-catalyzed cleavage of DNA, which has a temperature optimum between 40 "C and 45 'C [30,38], suggesting that either different mechanisms . Our cleavage experiments and the analysis of melting temperatures confirms that the EcoRI endonuclease is specific for double-stranded substrates as was deduced already by Greene et al [30] from the temperature dependence of the cleavage of d(pT-G-A-A-T-T-C-A) by the EcoRI endonuclease.…”

Section: Ecori Endonuclease and The Decadeoxynucleotides D(pg-g-g-a-amentioning

confidence: 86%

“…Oligodeoxynucleotides Ia and Ib are not cleaved at 45 "C. This result is not typical for the EcoRI-endonuclease-catalyzed cleavage of DNA, which has a temperature optimum between 40 "C and 45 'C [30,38], suggesting that either different mechanisms . Our cleavage experiments and the analysis of melting temperatures confirms that the EcoRI endonuclease is specific for double-stranded substrates as was deduced already by Greene et al [30] from the temperature dependence of the cleavage of d(pT-G-A-A-T-T-C-A) by the EcoRI endonuclease. By analogy it may be conjectured that the MspI endonuclease which was reported to cleave singlestranded oligodeoxynucleotides [35] is also specific for doublestranded substrates and interacts with these substrates as such rather than producing a duplex structure subsequent to binding of single strands to the enzyme.…”

Section: Ecori Endonuclease and The Decadeoxynucleotides D(pg-g-g-a-amentioning

confidence: 86%

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The influence of sequences adjacent to the recognition site on the cleavage of oligodeoxynucleotides by the EcoRI endonuclease

Alves

Pingoud

Haupt

et al. 1984

European Journal of Biochemistry

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We have investigated the influence of the nucleotide sequence adjacent to the recognition site on the rate of cleavage of DNA by the restriction endonuclease EcoRI. For this purpose two decadeoxynucleotides, (Ib) were synthesized. The duplex Ia . Ib is cleaved by EcoRI preferentially in the dA-rich strand (approximately 10 times over the dG-rich strand). The individual nucleotides Ia and Ib are also cleaved by EcoRI, Ib at a higher rate than Ia and both at a lower rate than Ia . Ib. The temperature dependence of the reaction rate shows that only double-stranded oligodeoxynucleotides are substrates for the EcoRI endonuclease. We have, furthermore, synthesized oligomers of d(G-G-A-A-T-T-C-C), which contain two, three and four EcoRI sites, respectively. These oligodeoxynucleotides are preferentially cleaved at the sites next to the 5' end, where the recognition site is only flanked by one dG.dC base pair, in contrast to the other sites which are flanked by three such pairs. These data indicate that sequences adjacent to the recognition site influence the rate of cleavage: dA'dT base pairs enhance and dG.dC base pairs slow down the hydrolytic activity of the EcoRI endonuclease. d(G-G-G-A-A-T-T-C-T-T) (Ia) and d(A-A-G-A-A-T-T-C-C-C)The class I1 restriction endonuclease EcoRI recognizes the DNA sequence [ I ] and cleaves the DNA with extreme specificity within this sequence as indicated (for recent reviews cf. [2-41). Although the occurrence of the recognition sequence seems to be sufficient for double-strand scission, the rate of cleavage has been observed to be dependent on additional structural features of the DNA. This result was first reported for bacteriophage A DNA by Thomas and Davis [5] and investigated in more detail later also by Halford et al. [6] and Berkner and Folk [7], who showed that the five EcoRI sites in bacteriophage A DNA are cleaved by the EcoRI endonuclease with different rates. Similar results were obtained later by Goldstein et al. [8] with bacteriophage T4 DNA and by Forsblom et al. [9] with adenovirus DNA. Rubin and Modrich [lo] have pointed out that the differences in the rate of doublestrand cleavage may be related to the differences observed in the rate of accumulation of the intermediate containing a single-strand break. The main conclusion that can be drawn from these investigations is that the substrate dependence of the rate of reaction most likely is a consequence of sequence differences next to the EcoRI sites of the substrates. Preferential cleavage is also observed at EcoR* sites [11,12]: among the many EcoR* sites in 4x174 (RF) DNA five are cleaved rapidly, while others with the same hexanucleotide sequence are refractory to cleavage.In order to find out in what manner the interaction of the EcoRI endonuclease with its substrate is modulated by the sequences flanking the EcoRI site, we have synthesized Enzyme. Restriction endonuclease EcoRI (EC 3.1.23.1 3). different oligodeoxynucleotides which contain the EcoRI site. Using oligodeoxynucleotides rather than macromolecula...

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Section: Ecori Endonuclease and The Decadeoxynucleotides D(pg-g-g-a-amentioning

confidence: 86%

Section: Ecori Endonuclease and The Decadeoxynucleotides D(pg-g-g-a-amentioning

confidence: 86%

The influence of sequences adjacent to the recognition site on the cleavage of oligodeoxynucleotides by the EcoRI endonuclease

Alves

Pingoud

Haupt

et al. 1984

European Journal of Biochemistry

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“…A recent statistical analysis of tumours occuring in patients over 50 years old suggested that the tumours studied, and their numbers, could be related to a decrease in the integrity of the genome (Dix et al, 1980 (Rubery & Newton, 1973;Browne & Burdon, 1977;Greene et al, 1975;DeWichter et al, 1971;Berneman et al, 1978). Groudine et al (1981) (Breznik & Cohen, 1982).…”

Section: -Mc Depletion During Tumour Developmentmentioning

confidence: 99%

5-Methylcytosine depletion during tumour development: An extension of the miscoding concept

Nyce¹,

Weinhouse²,

Magee³

1983

Br J Cancer

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Perhaps the most ubiquitous aspect of tumours and the neoplastic cells of which they are composed is their loss of the ability to control cellular functions in a normal way. During the development of the malignant state, the neoplastic cell becomes increasingly refractory to both the internal and external stimulae which integrate its normal counterparts into functional tissues and organ systems. This lack of integration is associated with alterations in the expression of a host of gene products, including, for example, the ectopic biosynthesis of hormones (

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“…The complete (95-100%) inhibition of the reaction was reached at 5 mM THP(A). The higher concentration of THP(A) needed for complete inhibition of the oligonucleotide cleavage (3-5-fold) as compared with plasmid DNA could be attributed to the different reaction conditions employed in the two experiments, different flanking sequence of recognition sites, and known differences in the reaction kinetics of restriction enzymes on short duplexes of 8 -12 bp compared with those on longer DNA (35).…”

Section: Effect Of Thps and Osmolytes On Dna Cleavage By Ecorimentioning

confidence: 99%

Effect of Tetrahydropyrimidine Derivatives on Protein-Nucleic Acids Interaction

Malin¹,

Iakobashvili

Lapidot

1999

Journal of Biological Chemistry

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2-Methyl-4-carboxy,5-hydroxy-3,4,5,6-tetrahydropyrimidine (THP(A) or hydroxyectoine) and 2-methyl,4-carboxy-3,4,5,6-tetrahydropyrimidine (THP(B) or ectoine) are now recognized as ubiquitous bacterial osmoprotectants. To evaluate the impact of tetrahydropyrimidine derivatives (THPs) on protein-DNA interaction and on restriction-modification systems, we have examined their effect on the cleavage of plasmid DNA by 10 type II restriction endonucleases. THP(A) completely arrested the cleavage of plasmid and bacteriophage DNA by EcoRI endonuclease at 0.4 mM and the oligonucleotide (d(CGCGAATTCGCG)) 2 at about 4.0 mM. THP(B) was 10-fold less effective than THP(A), whereas for betaine and proline, a notable inhibition was observed only at 100 mM. Similar effects of THP(A) were observed for all tested restriction endonucleases, except for SmaI and PvuII, which were inhibited only partially at 50 mM THP(A). No effect of THP(A) on the activity of DNase I, RNase A, and Taq DNA polymerase was noticed. Gelshift assays showed that THP(A) inhibited the EcoRI-(d-(CGCGAATTCGCG)) 2 complex formation, whereas facilitated diffusion of EcoRI along the DNA was not affected. Methylation of the carboxy group significantly decreased the activity of THPs, suggesting that their zwitterionic character is essential for the inhibition effect. Possible mechanisms of inhibition, the role of THPs in the modulation of the protein-DNA interaction, and the in vivo relevance of the observed phenomena are discussed.Two tetrahydropyrimidine derivatives identified in Streptomyces bacteria (1-3), one a previously unknown metabolite, THP(A), 1 and the other previously identified (as ectoine) in halophilic bacteria (4), THP(B), are now recognized as widely spread osmoprotectants within the bacterial world (5). The role and activities of THPs are of special interest as they represent a limited group of osmoprotectants that are synthesized de novo, in the bacterial cell, in contrast to those transported from the medium (6). Their synthesis in a number of Streptomyces strains as a response to increased salinity and elevated temperature was recently described (7). THPs are small molecules, highly soluble in water and neutral at physiological pH. NMR and x-ray crystallography data show that THPs are zwitterionic molecules with a delocalized charge in the NCN group (Fig. 1) and form the half-chair conformation (8).More information has been accumulated lately on THPs activity in living cells. It was found that exogenously provided ectoine (THP(B)) could reverse growth inhibition, caused by osmotic stress, in Escherichia coli (9), Corynebacterium glutamicum (10), and the soil bacterium Rhizobium meliloti (11). We demonstrated that exogenously provided THP(A), like THP(B), reversed inhibition of E. coli growth by osmotic stress, and moreover, both THP(A) and THP(B) could stimulate growth of E. coli at an elevated temperature (43°C) (7). Recently cloned genes for ectoine synthesis from Halomonas elongata (12) and from Marinococcus halophilus (13) were demonstrated ...

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Restriction and modification of a self-complementary octanucleotide containing the EcoRI substrate

Cited by 161 publications

References 22 publications

The influence of sequences adjacent to the recognition site on the cleavage of oligodeoxynucleotides by the EcoRI endonuclease

The influence of sequences adjacent to the recognition site on the cleavage of oligodeoxynucleotides by the EcoRI endonuclease

5-Methylcytosine depletion during tumour development: An extension of the miscoding concept

Effect of Tetrahydropyrimidine Derivatives on Protein-Nucleic Acids Interaction

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