Interaction of EcoRII restriction and modification enzyme with synthetic DNA fragments. IV. DNA duplexes with phosphoamide and pyrophosphate internucleotide bonds--the substrates for the study of single-strand breaks

A set of DNA duplexes with repeated EcoRII, EcoRI and AluI restriction endonuclease recognition sites in which EcoRII scissile phosphodiester bonds were replaced by phosphoramide or uncleavable pyrophosphate bonds have been synthesized. Endonuclease EcoRII was found not to cleave the substrate at the phosphoramide bond. The substrates containing non-nydrolysable pyrophosphate or phosphoramide bonds in one of the chains of EcoRII recognition sites were used to show that this enzyme is able to catalyze single-strand scissions. These scissions occur both in dA- and dT-containing chains of the recognition site. Endonuclease EcoRII interacts with both strands of the DNA recognition site, each of them being cleaved independently on the cleavage of the other. Synthesized DNA-duplexes are cleaved specifically by EcoRI and AluI endonucleases, this cleavage being retarded if the modified bonds are in the recognition site (EcoRI) or flank it (AluI). For EcoRII and AluI this effect is more pronounced in the case of substrates with pyrophosphate bonds than with the phosphoramide ones.