Efficiency of the cleavage of DNA duplexes with one recognition site by EcoRII restriction endonuclease decreases with the increase in substrate length. DNA duplexes more than 215 base pairs long are practically not cleaved by this enzyme. It has been found that in the presence of substrates 11-14 base pairs long acceleration of hydrolysis of extended single-site substrates by EcoRII enzyme is observed. The level of hydrolysis stimulation is dependent on the length and concentration of the second substrate. A model system for the study of the molecular and kinetic mechanism of EcoRII endonuclease stimulation has been proposed, including a 30-membered single-site substrate and DNA duplexes, modified at heterocyclic bases and internucleotide phosphate groups in the recognition site, as activators. The modified DNA duplexes can activate hydrolysis of the 30-membered substrate and phage T3 DNA. Their influence on the cleavage of extended substrates is dependent on the type of modification and its localization in the recognition site. It has been demonstrated that EcoRII endonuclease carries out cooperative interaction with two recognition sites in DNA.

Cleavage by restriction endonucleases MvaI kappa EcoRII of DNA duplexes, in which the internal deoxycytidine in one of the strands of the recognition site is substituted for 5-fluorodeoxycytidine, has been studied. It has been found that the modified strands of these duplexes are scissed by endonuclease MvaI with a greater efficiency than the intact ones. Endonuclease EcoRII does not practically cleave such substrate analogs. The UV absorbance spectra of 5-fluorodeoxycytidine and the 14-member oligodeoxyribonucleotide with modification in the middle of the strand have been measured. A hypothesis has been put forward about the structural features of 5-fluorodeoxycytidine-containing DNA duplexes.

The binding of restriction endonuclease EcoRII to synthetic oligodeoxyribonucleotide substrates 11 to 30 bp long was investigated by gradient polyacrylamide gel electrophoresis under nondenaturing conditions in the absence of Mg2+ ions. Irrespective of the substrate's length, two types of specific DNA-protein complexes were shown to be formed. Their mobility in gel was close to that of the monomer and the dimer of the marker ovalbumin. The number of such complexes in solution depended on the ratio of the molar concentrations of restriction endonuclease EcoRII and the DNA duplex. The possible structure of the complexes is discussed.

All DNA (cytosine-5)-methyltransferases contain a single conserved cysteine. It has been proposed that this cysteine initiates catalysis by attacking the C6 of cytosine and thereby activating the normally inert C5 position. We show here that substitutions of this cysteine in the E. coli methylase M. EcoRII with either serine or tryptophan results in a complete loss of ability to transfer methyl groups to DNA. Interestingly, mutants with either serine or glycine substitution bind tightly to substrate DNA. These mutants resemble the wild-type enzyme in that their binding to substrate is not eliminated by the presence of non-specific DNA in the reaction, it is sensitive to methylation status of the substrate and is stimulated by an analog of the methyl donor. Hence the conserved cysteine is not essential for the specific stable binding of the enzyme to its substrate. However, substitution of the cysteine with the bulkier tryptophan does reduce DNA binding. We also report here a novel procedure for the synthesis of DNA containing 5-fluorocytosine. Further, we show that a DNA substrate for M. EcoRII in which the target cytosine is replaced by 5-fluorocytosine is a mechanism-based inhibitor of the enzyme and that it forms an irreversible complex with the enzyme. As expected, this modified substrate does not form irreversible complexes with the mutants.

Binding of EcoRII restriction endonuclease to synthetic oligodeoxyribonucleotide substrates of 11-30 base pairs long was investigated by polyacrylamide gel electrophoresis under nondenaturing conditions in the absence of Mg2+ ions. Irrespective of the length of a substrate, two types of specific DNA-protein complexes were shown to be formed. Their mobility in gel was close to that of the monomer (45 kDa) and dimer (90 kDa) of marker protein, ovalbumin. The ratio of these complexes in solution depended on that of the molar concentrations of EcoRII restriction endonuclease and DNA duplexes. The possible structure of the complexes is discussed.

The cleavage of synthetic DNA duplexes containing 1,3-propanediol, 1,2-dideoxy-D-ribofuranose or 9-[1'-hydroxy-2'-(hydroxymethyl)ethoxy]methylguanine (glG) residues instead of one of dG residues or one of the nucleosides of the central base pair of the recognition site by SsoII restriction endonuclease (decreases CCNGG) has been studied. It is found that the non-nucleotide insertions (except for glG) result in a change of the SsoII cleavage site and an increase of the efficiency of the cleavage. The novel noncanonical cleavage occurs at the phosphodiester bond adjoining the non-nucleotide insert from the 5'-end.

Phosphoramidite derivative of 9-[1'-hydroxy-2'-(hydroxymethyl)ethoxy]methylguanine (glG) is synthesized which allows one to introduce point modifications in any position of the chemically prepared oligonucleotide chain. Oligonucleotides with 5'-terminal glG can be used in chemical ligation promoted by cyanogen bromide. The modified oligonucleotide duplexes were characterized by melting curves and CD spectra.

A spectrophotometric method for continuous monitoring the cleavage of DNA duplexes by type II restriction endonucleases was proposed. The time course of cleavage of a 14-membered DNA duplex by MvaI endonuclease was obtained. The spectrophotometric method is characterized by rapidity and high precision in determining the kinetic parameters of the reaction. It can be recommended for testing the preparations for the presence of restriction endonucleases, rapid determination of the activity of any restriction endonucleases, highly precise quantitative analysis of the restriction enzyme catalysed reactions.

The interaction of enzymes SsoII (decreases CCNGG) and MvaI (CC decreases A/TGG) with concatemeric DNA duplexes used earlier to study EcoRII (decreases CCA/TGG) TGG was investigated with a view of elucidating the general principles of the restriction endonuclease function. A pattern common for all the three enzymes was observed with DNA duplexes containing AA or TT pairs in the central position of the recognition site. The AA pair blocks or substantially hinders the endonuclease action, whereas the TT pair is either less inhibitory or altogether inert. SsoII, similar to EcoRII was able to processively cleave the concatemeric substrates and to interact with (or to be close to) the hydrogen in the 5th position of the outer dC residue of the recognition site. MvaI was found to differ from EcoRII in the way they recognize and cleave the same nucleotide sequence. The substrate-bound MvaI molecule is incapable of linear diffusion along the DNA. Effective hydrolysis of dU- and m5dC-containing polymers rules out the participation of hydrophobic contacts of the enzyme with the methyl group of the dT residue and with the 5th hydrogen of the outer dC residue of the recognition site in DNA-protein interactions.

Phosphoramidite derivative of 9-[1'-hydroxy-2'-(hydroxymethyl)ethoxy]methylguanine (glG) is synthesized which allows one to introduce point modifications in any position of the chemically prepared oligonucleotide chain. Oligonucleotides with 5'-terminal glG can be used in chemical ligation promoted by cyanogen bromide. The modified oligonucleotide duplexes were characterized by melting curves and CD spectra.

To study the interaction of the restriction endonucleases Mval and Eco RII with DNA we have synthesized some modified oligonucleotides. The results of hydrolysis demonstrate that both enzymes cleave their substrate by different mechanism.

DNA duplexes 14 bp long containing an EcoRII and MvaI restriction site in which a nucleoside is substituted by 1,3-diaminopropane or 1,3-propanediol residue have been chemically synthesized. Diaminopropane bridge was introduced by the chemical ligation, whereas the oligonucleotide containing propanediol was prepared by automatic solid phase phosphoroamidite method on "Victoria-4M" synthesizer. As CD and UV spectra show, the modification destabilises the duplex by 18-20 degrees C without essential distortion of the double helix, except for increase of the conformational mobility in the modified site.

The interaction of MvaI restriction endonuclease with 14-membered deoxyribonucleotide duplexes containing modifications within the recognition site (CCA/TGG) has been studied. Substitution of m5dC for the internal dC residue, as well as substitution of fl5dU or rU for dT did not influence the initial rate of hydrolysis (v0) 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.

The cleavage of synthetic DNA duplexes by the restriction endonuclease MvaI has been studied. The main result of the cleavage experiments is that MvaI cleaves unmodified duplexes in two single strand scissions in separate events and that the two strands are cleaved at significantly different rates. One strand nicks within the recognition site do not affect the cleavage. Furthermore, neither a pyrophosphate internucleotide bond modification in one strand nor the absence of one phosphate group at the central dA-residue of the recognition site do inhibit the cleavage of the second strand.

Chemischer InformationsdienstVolume 16, Issue 5 Natural Products ChemInform Abstract: CHEMICAL SYNTHESIS OF NONADEOXYRIBONUCLEOTIDES CONTAINING MODIFIED URACIL, 5-BROMOURACIL AND 5-METHYLCYTOSINE BASES BY THE TRIESTER METHOD A. ROSENTHAL, A. ROSENTHALSearch for more papers by this authorD. CECH, D. CECHSearch for more papers by this authorV. P. VEIKO, V. P. VEIKOSearch for more papers by this authorI. S. OREZKAJA, I. S. OREZKAJASearch for more papers by this authorE. A. ROMANOVA, E. A. ROMANOVASearch for more papers by this authorA. A. ELOV, A. A. ELOVSearch for more papers by this authorV. G. METELEV, V. G. METELEVSearch for more papers by this authorE. S. GROMOVA, E. S. GROMOVASearch for more papers by this authorZ. A. SHABAROVA, Z. A. SHABAROVASearch for more papers by this author A. ROSENTHAL, A. ROSENTHALSearch for more papers by this authorD. CECH, D. CECHSearch for more papers by this authorV. P. VEIKO, V. P. VEIKOSearch for more papers by this authorI. S. OREZKAJA, I. S. OREZKAJASearch for more papers by this authorE. A. ROMANOVA, E. A. ROMANOVASearch for more papers by this authorA. A. ELOV, A. A. ELOVSearch for more papers by this authorV. G. METELEV, V. G. METELEVSearch for more papers by this authorE. S. GROMOVA, E. S. GROMOVASearch for more papers by this authorZ. A. SHABAROVA, Z. A. SHABAROVASearch for more papers by this author First published: February 5, 1985 https://doi.org/10.1002/chin.198505344Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume16, Issue5February 5, 1985 RelatedInformation

In order to investigate the interaction of Eco RII restriction and modification enzymes with synthetic DNA fragments three nonadeoxyribonucleotides containing the modified bases uracil, 5-bromouracil and 5-methylcytosine were synthesized according to the phosphate tri-ester approach using TPS/l-methylimidazole as the condensation agent. The patterns of these modified DNA fragments obtained by Maxam/Gilbert sequence technique are presented.

Interaction of EcoRII restriction endonuclease with a set of synthetic concatemer DNA duplexes with natural and modified sites for this enzyme has been studied. DNA duplexes with repeated natural sites are cleaved by EcoRII. Substitution of central AT‐pair in the recognition site for a non‐complementary TT‐ or AA‐pair reduces the rate of cleavage, this effect being much more pronounced in the last case. Absence of site flanking in one strand from the 5′‐terminus also results in very slow cleavage. The results obtained testify to the interaction of EcoRII with both strands of the substrate.

In order to investigate the interaction of Eco RII restriction and modification enzymes with synthetic DNA fragments three nonadeoxyribonucleotides containing the modified bases uracil, 5-bromouracil and 5-methylcytosine were synthesized according to the phosphate tri-ester approach using TPS/l-methylimidazole as the condensation agent. The patterns of these modified DNA fragments obtained by Maxam/Gilbert sequence technique are presented.