Apoptotic DNA degradation: Evidence for novel enzymes

Apoptosis is characterized by multiple morphological and biochemical changes. One biochemical change that has been primarily associated with apoptosis is the cleavage of chromatin in the internucleosomal regions. We have taken two independent approaches to investigating the enzyme(s) responsible for such cleavage. First, using SDS-PAGE gels with 32P-labelled DNA incorporated into the matrix, we identified a nuclease activity (termed NUC18) from apoptotic thymocytes. This enzyme has been purified to homogeneity and the activity of the pure protein is dependent on Ca2+ and Mg2+ while inhibited by Zn2+ and aurintricarboxylic acid. This protein is found in the nucleus of apoptotic and nonapoptotic cells but is maintained in nondying cells in a large-molecular-weight inactive complex. NUC18 has a denatured molecular weight of 18 Kd but elutes from gel filtration columns with a native molecular weight of ≃ 25 Kd. Although an exhaustive search has not been performed, NUC18 has been identified in several cell lines and tissues. Our second approach is designed specifically to detect internucleosomal cleavage of DNA, an obvious requirement for an apoptotic nuclease. By examining the degradation of HeLa chromatin, we have identified a low-molecular-weight (≈ 23 Kd native molecular weight) internucleosomal cleavage enzyme active in nuclear extracts from glucocorticoid-treated thymocytes. This activity is also dependent upon Ca2+ and Mg2+ and is inhibited by Zn2+ as well as aurintricarboxylic acid. It is present in a variety of cell lines and tissues and is maintained in control cells in a latent state prior to apoptosis. In addition to similarities in physical properties, the two enzymes appear to be immunologically related to one another by virtue of their ability to interact with the same antibody. Overall, using independent approaches, we have identified two nucleases with similar biochemical properties whose activity correlates with apoptosis. The current work suggests that these are novel and perhaps closely related enzymes.

Duke Scholars

Author Monty Hughes Jr. Urology