PURIFICATION AND PROPERTIES OF DNASE-GAMMA FROM APOPTOTIC RAT THYMOCYTES

We previously identified three distinct DNA endonucleases, DNases alph a, beta and gamma, present in rat thymocyte nuclei. On the basis of th eir enzymic and biochemical properties, gamma-type DNase was regarded as a candidate for the apoptotic endonuclease. Here we purified DNase ?, to apparent homogeneity from apoptotic rat thymocyte nuclei induced by X-irradiation and characterized its properties in detail. The puri fied DNase gamma exhibited one predominant protein band on SDS/PAGE an d an endonuclease activity in a zymography with an estimated molecular mass of 33 kDa. The molecular mass of the native form determined by G 2000SW gel-filtration HPLC was 30 kDa. Amino acid analysis showed that the amino acid composition of DNase gamma was similar to that of rat DNase I (molecular mass 32 kDa) but different with regard to alanine a nd lysine residues, The N-terminal amino acid sequence of DNase gamma was revealed to be not identical with that of rat DNase I, In accordan ce with previous studies, homogeneously purified DNase gamma requires both Ca2+ and Mg2+ for activity. This requirement could be partially s upplied by Mn2+. Of the bivalent metal ions tested, Co2+, Ni2+, Cu2+ a nd Zn2+ inhibited DNase gamma activity. These bivalent cations also su ppressed apoptotic DNA fragmentation in rat thymocytes irradiated by X -rays. The same order of inhibitory ability was observed for these biv alent metal ions in vivo (in intact cells) and in vitro, suggesting th at the suppression of apoptotic DNA fragmentation at the cellular leve l is due to the inhibition of DNase gamma. DNase gamma activity was fo und to exist at high levels in spleen, lymph node, thymus, liver and k idney, but little was present in brain, heart or pancreas. On the basi s of these findings, together with previous data, we conclude that DNa se gamma is a novel DNase I-like endonuclease responsible for internuc leosomal cleavage of chromatin during thymic apoptosis.