Relaxation of recognition sequence of specific endonuclease HlndIII

Under the standard reaction conditions, the restriction endonuclease HindIII cleaves double-stranded DNA, within the recognition sequence--A/AGCTT--at the position indicated by the arrow. In the presence of dimethyl sulfoxide the substrate specificity of this enzyme is reduced and cleavages occur at additional sites. We have determined the secondary sites in pBR322 DNA recognized by HindIII endonuclease under relaxed conditions and found that it cleaves the hexanucleotides: G/AGCTT, A/GGCTT, A/TGCTT, A/ATCTT, A/AGCCT, A/AGCAT, A/AGCGT, A/AGCTC, at the positions indicated by the arrows, producing fragments with cohesive ends.