Conversion of bovine pancreatic DNase I to a repair endonuclease with a high selectivity for abasic sites

Bovine pancreatic deoxyribonncleaae I(DNase I) is a nuclease of relatively low specificity which interacts with DNA in the minor groove. No contacts a re made between the protein and the major groove of the nucleic acid. DNase I is structurally homologous to exonuclease III, a DNA-repair enzyme with multiple activities. One of the main differences between the two enzymes is the presence of an additional alpha-helix in exonuclease III, in a positio n suggestive of interaction with the major groove of DNA, Recombinant DNA t echniques have been used to add 14 amino acids, comprising the 10 amino aci ds of the exonuclease III alpha-helix flanked by a glycine rich region, to DNase I. The polypeptide has been inserted after serine 174, an amino acid on the surface of DNase I corresponding to the location of the extra alpha- helix in exonuclease III, The recombinant protein, DNase-exohelix, has been purified and its catalytic activities towards DNA investigated. The recomb inant protein demonstrated a high selectivity for endonucleolytic cleavage at abasic sites in DNA, a property of exonuclease III but not native DNase I. Thus the insertion of 14 amino acids at Ser174, converts DNase I to an e xonuclease III-like enzyme with DNA-repair properties.