NONENZYMATIC CLEAVAGE AND LIGATION OF DNA AT A 3 A.T BASE-PAIR SITE -A 2-STEP PSEUDOHYDROLYSIS OF DNA

The chemical nuclease Mn-TMPyP/KHSO5 is able to hydroxylate 5' carbon- hydrogen bonds of deoxyribose units at three contiguous A.T base pairs sites and initiate DNA rupture. Double-strand cleavage sites consist of trinucleotide sequences with a four nucleotide 3' stagger of the cl eaved residues. Both strand nicks are identical and consist of 3'-phos phate termini facing a 5'-aldehyde residue which is easily reduced to a 5'-alcohol by NaBH4. These two successive treatments (oxidation plus reduction) are equivalent to a hydrolysis of the phosphodiester bond. Such mechanism is reminiscent of DNA restriction enzymes but with 5'- OH and 3'-phosphate ends at the site of cleavage. As demonstrated on d ouble-stranded ODN containing an (A.T)3 site, the chemically cleaved D NA fragments could be religated by a BrCN chemical method to re-form t he starting covalently linked DNA strand. Single- and double-strand li gations were obtained with yields ranging from 30 to 85%. Integrity of the re-formed double-stranded ODNs has been controlled (i) by quantit ative analysis of nucleosides released after enzymatic digestion and ( ii) by using a religated ODN as a substrate for a restriction enzyme ( BglI).