Evidence that non-caspase proteases are required for chromatin degradationduring apoptosis

Chromatin degradation into oligonucleosomal and approximate to 30-50 Kb fra gments is a hallmark of apoptosis. Crude nuclear extract from apoptotic rat thymocytes is able to recapitulate both types of DNA fragmentation in an a ssay using HeLa cell nuclei as an exogenous substrate. Using size exclusion chromatography we have identified a novel activity( approximate to 260 Kd) that produces only approximate to 30-50 Kb DNA fragments, and a 25 Kd acti vity that generates both approximate to 30-50 Kb and oligonucleosomal fragm ents. Both activities produced DNA fragments with 3'-OH termini, are depend ent on Ca2+ and Mg2+ and are inhibited by N-ethyl-maleimide, sodium tetrath ionate, aurin-tricarboxylic acid and sodium chloride, similar to other nucl eases implicated in apoptosis. These activities were inhibited by the serin e protease inhibitors N-tosyl-L-phenylalanine chloromethyl ketone and Na-p- tosyl-L-lysine chloromethyl ketone, but not by the serine protease inhibito r diisopropyl fluorophosphate, or by calpain inhibitors I or II, or the cap sase inhibitors Ac-Asp-Glu-Val-Asp-aldehyde, Ac-TyrVal-Ala-Asp-aldehyde, or Z-Val-Ala-Asp-fluoromethyl ketone. Both activities were insensitive to pro tease inhibitors when extracts were incubated with naked linear DNA, indica ting the presence of both nuclease and protease activities in the preparati on. Together, these observations suggest the involvement of non-caspase pro teases in apoptosis which perhaps function by altering chromatin substructu re and exposing it to nucleolytic attack.