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- Title
Conversion of bovine pancreatic DNase I to a repair endonuclease with a high selectivity for abasic sites.
- Authors
Cal, Santiago; Kok Leong Tang; McGregor, Alistair; Connolly, Bernard A.
- Abstract
Bovine pancreatic deoxyribonuclease I (DNase I) is a nuclease of relatively low specificity which interacts with DNA in the minor groove. No contacts are 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 α-helix in exonuclease III, in a position suggestive of interaction with the major groove of DNA. Recombinant DNA techniques have been used to add 14 amino acids, comprising the 10 amino acids of the exonuclease III α-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 α-helix in exonuclease III. The recombinant protein, DNase—exohelix, has been purified and its catalytic activities towards DNA investigated. The recombinant 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 exonuclease III-like enzyme with DNArepair properties.
- Subjects
DEOXYRIBONUCLEASES; DNA; PROTEINS; NUCLEIC acids; ENZYMES; AMINO acids
- Publication
EMBO Journal, 1998, Vol 17, Issue 23, p7128
- ISSN
0261-4189
- Publication type
Article
- DOI
10.1093/emboj/17.23.7128