Interaction of EcoRII restriction and modification enzymes with synthetic DNA fragments. VI. The binding and cleavage of substrates containing nucleotide analogs

Abstract

The present study deals with the binding and cleavage by EcoRII endonuclease of concatemer DNA duplexes containing EcoRII recognition sites (formula; see text) in which dT is replaced by dU or 5-bromodeoxyuridine, or 5'-terminal dC in the dT-containing strand is methylated at position 5. The enzyme molecule is found to interact with the methyl group of the dT residue of the DNA recognition site and to be at least in proximity to the H5 atom of the 5'-terminal dC residue in dT-containing strand of this site. Modification of any of these positions exerts an equal effects on the cleavage of both DNA strands. Endonuclease EcoRII was found to bind the substrate specifically. At the same time modification of the bases in recognized sequence may result in the formation of unproductive, though stable, enzyme-substrate complexes.

Sequence-dependent cleavage of mismatched DNA byBanI restriction endonuclease

“…Fortunately, such an activity can be usually avoided or minimized under normal buffer conditions (ie, those recommended by the manufacturers). However, previous reports and this study suggest that some restriction enzymes readily recognize and cleave mismatched DNA under normal conditions. In fact, mismatches in DNA can arise from many sources, such as misincorporation of nucleotides, deamination of mucleotide bases, and recombination or hybridization between homologous DNA sequences during both in vivo and in vitro processes.…”

“…Binding to the specific recognition site with suitable flanking sequences will then activate the restriction enzyme by increasing catalysis . Synthetic modified oligodeoxyribonucleotide duplexes have been applied to probe the DNA determinants required for such a recognition by a series of restriction enzymes . The success of this approach depends mostly on the availability of a large number of suitably modified DNA substrates.…”

“…Introduction of altered single base (mismatched substrate) or base pair (bp) (star substrate) within the recognition sequence can be used to investigate the accuracy of a restriction enzyme in discriminating between the canonical ( ca ) recognition sequence and its most closely related noncanonical ( nc ) sequence(s). The Eco RI, Eco RII, Eco RV, and Mva I restriction enzymes have been shown to cleave DNA substrates containing mismatches within their recognition sequences. In the case of Eco RII and Mva I, which recognize the same pentanucleotide palindromic sequence CCA/TGG but cleave it at different positions, the replacement of the central A/T (or T/A) bp with a T/T mismatch slightly decreased the hydrolysis by both enzymes, while the substitution of an A/A at the same position blocked the action of these enzymes to a larger extent.…”

“…The Eco RI, Eco RII, Eco RV, and Mva I restriction enzymes have been shown to cleave DNA substrates containing mismatches within their recognition sequences. In the case of Eco RII and Mva I, which recognize the same pentanucleotide palindromic sequence CCA/TGG but cleave it at different positions, the replacement of the central A/T (or T/A) bp with a T/T mismatch slightly decreased the hydrolysis by both enzymes, while the substitution of an A/A at the same position blocked the action of these enzymes to a larger extent. However, owing to the fact that these studies aimed mainly at understanding the influence of various base modifications, removal or replacement of bps, on the function of Eco RII and Mva I, detailed information from DNA substrates containing various mismatched bases at each site of a recognition sequence was not available.…”

Interaction of the Restriction Endonuclease EcoRV with the Deoxyguanosine and Deoxycytidine Bases in Its Recognition Sequence

“…This method has been widely used to study a variety of DNA-binding proteins (Yansura et al, 1979;Goedell et al, 1978;Fisher & Caruthers, 1979) and several enzymes that act upon DNA especially restriction endonucleases (Dwyer-Hallquist et al, 1982;Ono et al, 1984;Yolov et al, 1985;Seela & Driller, 1986;Jiriciny et al, 1986;Brennan et al, 1986a,b;McLaughlin et al, 1987;Seela & Kehne, 1987;Ono & Ueda, 1987;Hayakawa et al, 1988;Lesser et al, 1990Lesser et al, , 1993Zebala et al, 1992b). This technique consists of preparing a base analogue in which a group or atom that has the potential to interact with a DNA-binding protein (Seeman et al, 1976) is deleted.…”