Conversion of bovine pancreatic DNase I to a repair endonuclease with a high selectivity for abasic sites.
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 alpha-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 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 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 DNA-repair properties.
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Department of Biochemistry and Genetics, The University of Newcastle, Newcastle upon Tyne NE2 4HH, UK.
Tan
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KL
McGregor
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Connolly
B A
BA
D000818:Animals; D001483:Base Sequence; D019757:Carbon-Oxygen Lyases / Q000378:metabolism; D002417:Cattle; D004247:DNA; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D003850:Deoxyribonuclease I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D043223:Deoxyribonuclease IV (Phage T4-Induced); D005090:Exodeoxyribonucleases / Q000378:metabolism*; D005092:Exonucleases / Q000378:metabolism; D015870:Gene Expression; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016914:Ribonuclease H / Q000378:metabolism; D012694:Serine; D014443:Tyrosine
D014443:Tyrosine; D012694:Serine; D004247:DNA; D005090:Exodeoxyribonucleases; D005092:Exonucleases; C020133:exodeoxyribonuclease III; C022650:spleen exonuclease; D003850:Deoxyribonuclease I; D043223:Deoxyribonuclease IV (Phage T4-Induced); D016914:Ribonuclease H; D019757:Carbon-Oxygen Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase