Interaction of EcoRII restriction and modification enzymes with synthetic DNA fragments. V. Study of single-strand cleavages

Yolov, A.A.; Громова, Е. С.; Кубарева, Е. А.; Potapov, V. K.; Shabarova, Zoe A.

doi:10.1093/nar/13.24.8969

Cited by 19 publications

(22 citation statements)

References 12 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…1. The disappearance of high-mobility complex B 0 strands of the substrate, respectively (1,11,12). c The concentrations of EcoRII monomers to deoxyoligonucleotides were 1:1 for the duplexes I±IX, and also 1:5 for duplexes IV±VII.…”

Section: Binding Of 14-bp Canonical Dna Duplex I To Ecoriimentioning

confidence: 99%

A Model of EcoRII Restriction Endonuclease Action: The Active Complex is Most Likely Formed by One Protein Subunit and One DNA Recognition Site

1999

View full text Add to dashboard Cite

SummaryTo elucidate the mechanism of interaction of restriction endonuclease EcoRII with DNA, we studied by native gel electrophoresis the binding of this endonuclease to a set of synthetic DNAduplexescontaining the modi® ed or canonical recognition sequence Keywords EcoRII restriction endonuclease; enzyme±substrate complexes; mechanism of enzyme action; modi® ed DNA substrates; nondenaturing polyacrylamide gel assay.

show abstract

Section: Binding Of 14-bp Canonical Dna Duplex I To Ecoriimentioning

confidence: 99%

A Model of EcoRII Restriction Endonuclease Action: The Active Complex is Most Likely Formed by One Protein Subunit and One DNA Recognition Site

1999

View full text Add to dashboard Cite

show abstract

“…Very different data were obtained for EcoRII endonuclease. Here, the substitution f a pyrophosphate bond for the scissile phosphodiester bond in one strand leads to a remarkable decrease in the rate of cleavage of the unmodified strand [8]. Therefore, the introduction of an additional phosphate group within the recognition site of MvuI endonuclease does not influence the interaction of that enzyme with DNA.…”

Section: Digestion Of Substrates Which Are Modified At the Central Bamentioning

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

View full text Add to dashboard Cite

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.

show abstract

“…The products of the cleavage of 32P-labelled DNA duplexes were analyzed on 12% or 20% polyacrylamide gels containing 7 M urea. The radioactivity of the gel slices was…”

Section: Analysis Of Cleavage Productsmentioning

confidence: 99%

“…Duplex VII (n > 20 bp) has a canonical EcoRII site repeated every 9 bp, and duplex VIIa has pyrophosphate bonds which replace hydrolyzable phosphodiester bonds [7]. Duplex VIIa is capable of specific binding to EcoRII [ll].…”

Section: Fig 3 a Scheme Of The Ecoriilsubstrate Interactionmentioning

confidence: 99%

“…Duplex VIIa is capable of specific binding to EcoRII [ll]. The affinities of duplexes VII and VIIa for EcoRII are nearly equal, but the duplex VIIa is not cleaved by the enzyme [7,11]. The study of the activation has revealed that the unmodified duplex VII initially inhibits the hydrolysis of DNA-duplex I and stimulates its cleavage (Fig.…”

Section: Fig 3 a Scheme Of The Ecoriilsubstrate Interactionmentioning

confidence: 99%

See 1 more Smart Citation

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

Petrauskene

Кубарева

Gromova

et al. 1992

European Journal of Biochemistry

View full text Add to dashboard Cite

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...

show abstract

Interaction of EcoRII restriction and modification enzymes with synthetic DNA fragments. V. Study of single-strand cleavages

Abstract: Concatemer DNA duplexes which contain at the EcoRII restric-

Cited by 19 publications

References 12 publications

A Model of EcoRII Restriction Endonuclease Action: The Active Complex is Most Likely Formed by One Protein Subunit and One DNA Recognition Site

A Model of EcoRII Restriction Endonuclease Action: The Active Complex is Most Likely Formed by One Protein Subunit and One DNA Recognition Site

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

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

Contact Info

Product

Resources

About