Mechanism of action of Micrococcus luteus .gamma.-endonuclease

Abstract

Micrococcus luteus extracts contain .gamma.-endonuclease, a Mg2+-independent endonuclease that cleaves .gamma.-irradiated DNA. This enzyme has been purified approximately 1000-fold, and the purified enzyme was used to study its substrate specificity and mechanism of action. .gamma.-Endonuclease cleaves DNA containing either thymine glycols, urea residues, or apurinic sites but not undamaged DNA or DNA containing reduced apurinic sites. The enzyme has both N-glycosylase activity that releases thymine glycol residues from OsO4-treated DNA and an associated apurinic endonuclease activity. The location and nature of the cleavage site produced has been determined with DNA sequencing techniques. .gamma.-Endonculease cleaves DNA containing thymine glycols or apurinic sites immediately 3'' to the damaged or missing base. Cleavage results in a 5''-phosphate terminus and a 3'' baseless sugar residue. Cleavage sites can be converted to primers for DNA polymerase I by subsequent treatment with Escherichia coli exonuclease III. The mechanism of action of .gamma.-endonuclease and its substrate specificity are very similar to those identified for E. coli endonuclease III.