UTILIZATION OF AN IN-VITRO ASSAY TO EVALUATE CHROMATIN DEGRADATION BYCANDIDATE APOPTOTIC NUCLEASES

Apoptosis is commonly associated with the catabolism of the genome in the dying cell. The chromatin degradation occurs in essentially two fo rms: (1) internucleosomal DNA cleavage to generate oligonucleosomal-le ngth fragments (180-200 bp and multiples thereof), and (2) cleavage of higher order chromatin structures to generate approximate to 30-50 Kb fragments. To investigate this component of apoptosis and identify th e nuclease(s) responsible, we have developed and utilized an in vitro assay that recapitulates the genomic destruction seen during apoptosis in vivo and allows the simultaneous analysis of both forms of DNA deg radation from the same sample. Using this assay we evaluated the diges tion patterns of several candidate apoptotic nucleases: DNase I, DNase II, and cyclophilin (NUC18) as well as the bacterial enzyme micrococc al nuclease (not thought to be involved in apoptosis). Chromatin degra ded by DNase I formed a smear of DNA on conventional static-field agar ose gels and approximate to 30-50 Kb DNA fragments on pulsed field gel s. In contrast, DNase II, at a physiologically relevant pH, had no eff ect on the integrity of HeLa chromatin in either analysis. Similar to DNase I, cyclophilin C produced only approximate to 30-50 Kb DNA fragm ents but did not generate internucleosomal fragments. In contrast, mic rococcal nuclease generated both oligonucleosomal and approximate to 3 0-50 Kb DNA fragments. Nuclear extracts from glucocorticoid-treated ap optotic thymocytes generated oligonucleosomal DNA fragments and the la rger approximate to 30-50 Kb DNA fragments, fully recapitulating both types of apoptotic DNA degradation. Previously, differential sensitivi ty of nucleases to inhibition by Zn2+ was used to argue that two disti nct enzymes mediate approximate to 30-50 Kb DNA cleavage and internucl eosomal DNA degradation. While, the nuclease activity present in thymo cyte nuclear extracts was differentially sensitive to inhibition by Zn 2+ during short term incubations it was not during prolonged digestion s, suggesting that differences in DNA detection are likely to account for previous results. Together our studies show that none of the nucle ases commonly associated with apoptosis could fully recapitulate the D NA degradation seen in vivo.