Interaction between EcoRII restriction/modification enzymes and synthetic DNA fragments. Synthesis of substrates containing a single recognition site

Кузнецова, Светлана А.; Кубарева, Е. А.; Oretskaya, T. S.; Dolinnaya, Nina G.; Krynetskaya, N. F.; Gromova, E. S.; Shabarova, Zoe A.; Cech, Dieter

doi:10.7124/bc.0001fe

Cited by 3 publications

(4 citation statements)

References 11 publications

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“…Melting points of the substrates I, I1 and VI have been studied elsewhere [12]. The kinetic data of the digestion by MvaI endonuclease were determined both for the upper and the lower strand (indicated as T or A strand according to the central unit of the recognition site) of substrates I -VII (Table 1).…”

Section: Resultsmentioning

confidence: 99%

The role of modifications in oligonucleotides in sequence recognition by MvaI restriction endonuclease

Кубарева¹,

Pein²,

Gromova³

et al. 1988

European Journal of Biochemistry

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The interaction of MvaI restriction endonuclease with 14-membered deoxyribonucleotide duplexes containing modifications within the recognition site (CCA/TGG) has been studied. Substitution of m'dC for the internal dC residue, as well as substitution of fl'dU or rU for dT did not influence the initial rate of hydrolysis (vo) of modified strands, whereas the hydrolysis of unmodified strands was inhibited in some cases. Furthermore, the substitution of a pyrophosphate bond for a scissile phosphodiester bond in one strand completely inhibited digestion in this strand without any decrease of the rate of hydrolysis of the unmodified strand. In contrast to EcoRII endonuclease, which recognizes the same DNA sequence, in the case of MvaI endonuclease substrate recognition is possible in a wide range of conformational, electronic and hydrophobic alterations within the recognition site.

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Section: Resultsmentioning

confidence: 99%

The role of modifications in oligonucleotides in sequence recognition by MvaI restriction endonuclease

Кубарева¹,

Pein²,

Gromova³

et al. 1988

European Journal of Biochemistry

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“…Several of the DNA duplexes constructed (1, Id, l e , 2, 3-3c, 4-4c, 4f, 4h, 4m, 4n) have been characterized in this respect by UV spectroscopy and by circular dichroism (Dolinnaya et al, 1988;Gromova et al, 1984;Kubareva et al, 1990Kubareva et al, , 1991Kuznetsova et al, 1987bKuznetsova et al, , 1988. Summarizing these results with the data obtained from melting, circular dichroism, NMR and the X-ray structure of double-stranded oligonucleotides differing in primary structure from the compounds used here (Table 1) but carrying analogous point modifications (Delepierre et al, 1986;Delort et al, 1985;Diekmann and McLaughlin, 1988;Dolinnaya et al, 1986;Dolinnaya and Gryaznova, 1989;Frederick et al, 1988;Hunter et al, 1986;Kremer et al, 1987;Pieters et al, 1989;Purmal et al, 1984;Snowden-Ifft and Wemmer, 1990;Wang et al, 1982), one can arrive at the following conclusions.…”

Section: Structure and Stability Of Modified Substratesmentioning

confidence: 99%

“…Modifications not hindering complementary iteractions and not affecting the conformatinal mobility of individual fragments of a molecule (la-lc, 3a, 4a, 4c-4e, 4i and 41) do not reduce the thermal stability of duplexes (Dolinnaya et al, 1986(Dolinnaya et al, , 1988Kuznetsova et al, 1987b). Other modifications-mismatched base pairs (Id, l e , 3b, 3c, 4k) (Dolinnaya et al, 1988;Dolinnaya and Gryaznova, 1989;Gromova et al, 1984), nucleoside analogues m6A, m4C, I (4b, 4f-4h) (Kubareva et al, 1990), single-strand breaks, nucleotide deletions or non-nucleotide insertions (2a-2f, 4m, 4n) (Dolinnaya and Gryaznova, 1989;Kuznetsova et al, 1988;Pieters et al, 1989;Snowden-Ifft and Wemmer, 1990) lower the melting temperatures of the duplexes.…”

Section: Structure and Stability Of Modified Substratesmentioning

confidence: 99%

Peculiarities of recognition of CC^A/_TGG sequences in DNA by restriction endonucleases MvaI and EcoRII

Gromova

Кубарева

Vinogradova

et al. 1991

J of Molecular Recognition

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To elucidate the mechanism of action of restriction endonucleases MvaI and EcoRII a study was made of their interaction with a set of synthetic substrates in which the heterocyclic bases or the sugar-phosphate backbone had been modified; individual nucleotide residues had been removed or replaced with hydrocarbon bridges, and mismatched base pairs had been introduced. The groups of atoms in the heterocyclic bases and the phosphates in the recognition site that produce the most significant influence on the functioning of endonucleases MvaI and EcoRII were discerned. Profound differences were found in the functioning of the MvaI and EcoRII neoschizomers. The catalytic activity of EcoRII is significantly affected by any alteration in the recognition site structure and conformation, with a modification in one strand of the substrate causing the same decrease in the hydrolysis rate of both strands. Endonuclease MvaI is tolerant to a number of structural abnormalities; the latter sometimes affect only hydrolysis of one strand of the recognition site. The enzyme can preferentially cleave one of the substrate strands. Mismatched base pairs retard and sometimes block the hydrolysis. The effect depends on the particular enzyme, mismatch and its location.

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“…14 -30-bp substrates I -IV and IVa -1Ve were obtained by the triester method in solution or by the phosphoramidite method on a Victoria-4M automatic synthesizer (USSR) on a polymer support as described [9,101. Modified strands of substrates IVb and IVd were kindly given by Dr V. V. Butkus; the modified strand of substrate IVd was also synthesized by J. Backmann.…”

Section: Dna Duplexesmentioning

confidence: 99%

Mechanism of the interaction of EcoRII restriction endonuclease with two recognition sites

Petrauskene

Кубарева

Gromova

et al. 1992

European Journal of Biochemistry

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The efficiency of cleavage of DNA duplexes with single EcoRII recognition sites by the EcoRII restriction endonuclease decreases with increasing substrate length. DNA duplexes of more than 21 5 bp are not effectively cleaved by this enzyme. Acceleration of the hydrolysis of long single-site substrates by EcoRII is observed in the presence of 11 -14-bp substrates. The stimulation of hydrolysis depends on the length and concentration of the second substrate. To study the mechanism of EcoRII endonuclease stimulation, DNA duplexes with base analogs and modified internucleotide phosphate groups in the EcoRII site have been investigated as activators. These modified duplexes are cleaved by EcoRII enzyme with different effciences or are not cleaved at all. It has been discovered that the resistance of some of them can be overcome by incubation with a susceptible canonical substrate. The acceleration of cleavage of long single-site substrates depends on the type of modification of the activator. The modified DNA duplexes can activate EcoRII catalyzed hydrolysis if they can be cleaved by EcoRII themselves or in the presence of the second canonical substrate. It has been demonstrated that EcoRII endonuclease interacts in a cooperative way with two recognition sites in DNA. The cleavage of one of the recognition sites depends on the cleavage of the other. We suggest that the activator is not an allosteric effector but acts as a second substrate.EcoRII restriction endonuclease recognizes the (5')JCCiGG (3') sequence in DNA and cleaves it prior to the first nucleotide [l].It has been found recently [2, 31 that the mechanism of EcoRIIIDNA interaction is more complex than that of type-I1 restriction enzymes studied before [4, 51. This enzyme is not able to cleave phage T3 DNA with three EcoRII sites and phage T7 DNA with the unique EcoRII site (the single-site effect). Addition of susceptible DNA or short substrates may, however, stimulate such a cleavage. It was suggested that EcoRII requires at least two recognition sites for its activation [3]. In order to substantiate the mechanism of EcoRIIlsubstrate interaction, we describe here a useful model system to study the stimulation effect and to test a wide set of DNA duplexes containing modified and unmodified EcoRII sites as activators. The main purpose of using differently modified oligonucleotides was to determine the requirements for the activator: its structure and ability to be cleaved by EcoRII. Substrate analogs were chosen according to our recent findings that they are cleaved by EcoRII with different efCorrespondence to E. S. Gromova, A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow W-234, Russia, 119899Ahbveviations. m4C, N4-methyldeoxycytidine; m6A, N6-methyldeoxyadenosine; flsU, 5-fluorodeoxyuridine.Enzymes. Restriction endonucleases EcoRII and MvaI (EC 3.1.21.4); T4polynucleotide kinase (EC 2.7.1.78); T4 DNA ligase (EC 6.5.1.1). ficiencies or are not cleaved at all [6, 71. Additionally, the length of the substrates producing...

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Interaction between EcoRII restriction/modification enzymes and synthetic DNA fragments. Synthesis of substrates containing a single recognition site

Abstract: * Здесь и далее префикс d (дезокси) опущен, направление связи во всех дуплексах, как в дуплексе I.

Cited by 3 publications

References 11 publications

The role of modifications in oligonucleotides in sequence recognition by MvaI restriction endonuclease

The role of modifications in oligonucleotides in sequence recognition by MvaI restriction endonuclease

Peculiarities of recognition of CC^A/_TGG sequences in DNA by restriction endonucleases MvaI and EcoRII

Mechanism of the interaction of EcoRII restriction endonuclease with two recognition sites

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Interaction between EcoRII restriction/modification enzymes and synthetic DNA fragments. Synthesis of substrates containing a single recognition site

Abstract: * Здесь и далее префикс d (дезокси) опущен, направление связи во всех дуплексах, как в дуплексе I.

Cited by 3 publications

References 11 publications

The role of modifications in oligonucleotides in sequence recognition by MvaI restriction endonuclease

The role of modifications in oligonucleotides in sequence recognition by MvaI restriction endonuclease

Peculiarities of recognition of CCA/TGG sequences in DNA by restriction endonucleases MvaI and EcoRII

Mechanism of the interaction of EcoRII restriction endonuclease with two recognition sites

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Peculiarities of recognition of CC^A/_TGG sequences in DNA by restriction endonucleases MvaI and EcoRII