Micrococcus luteus endonucleases for apurinic/apyrimidinic sites in deoxyribonucleic acid. 1. Purification and general properties

Abstract

Two chromatographically distinct endonucleases from Micrococcus luteus, specific for apurinic and apyrimidinic sites (AP-endonucleases A and B), have been extensively purified and characterized. Both are free from DNA glycosylase, unspecific endonuclease, and phosphatase activities. The two enzymes behave as monomeric proteins of approximately 35000 daltons. In addition to their different chromatographic properties on CM-cellulose, P-cellulose, hydroxylapatite, and DNA--Sepharose, both AP-endonucleases can be distinguished as follows: AP-endonuclease A has an isoelectric point of 4.8, shows a half-life of 4 min at 45 degrees C, reacts optimally at pH 7.5 and has a KM value of 2.3 X 10(-6) M. AP-endonuclease B has a pI of 8.8, is more stable at 45 degrees C (half-life of 10 min), and reacts optimally between pH 6.5 and pH 8.5; its KM value is 3.7 X 10(-6) M.