The interaction of DNA duplexes containing 2-aminopurine with restriction endonucleases EcoRll and Ssoll

Abstract

Oligonucleotldes containing 2-amlnopurine (2-AP) In place of G or A in the recognition site of EcoRII (CCT/AGG) or Ssoll (CCNGG) restriction endonucleases have been synthesized In order to Investigate the specific interaction of DNA with these enzymes. Physicochemlcal properties (CD spectra and melting behaviour) have shown that DNA duplexes containing 2-aminopurine exist largely in a stable B-IIke form. 2-Aminopurine base paired with cytidine, however, essentially Influences the helix structure. The presence of a 2-APC mismatch strongly reduces the stability of the duplexes in comparison with the natural double strand, indicated by a blphaslc melting behaviour. Ssoil restriction endonuclease recognizes and cleaves the modified substrate with a 2-APT mismatch In the centre of the recognition site, but it does not cleave the duplexes containing 2-amlnopurine in place of inner and outer G, or both. EcoRII restriction endonuclease does not cleave duplexes containing 2-amlnopurine at all. The two-substrate mechanism of EcoRII-DNA interaction, however, allows hydrolysis of the duplex containing 2-amlnopurine In place of adenine In the presence of the canonical substrate. © 1995 Oxford University Press.