The role of topoisomerase II in the excision of DNA loop domains during apoptosis.

Disintegration of nuclear DNA into high molecular weight (HMW) and oligonucleosomal DNA fragments represents two major periodicities of DNA fragmentation during apoptosis. These are thought to originate from the excision of DNA loop domains and from the cleavage of nuclear DNA at the internucleosomal positions, respectively. In this report, we demonstrate that different apoptotic insults induced apoptosis in NB-2a neuroblastoma cells that was invariably accompanied by the formation of HMW DNA fragments of about 50-100 kb but proceeded either with or without oligonucleosomal DNA cleavage, depending on the type of apoptotic inducer. We demonstrate that differences in the pattern of DNA fragmentation were reproducible in a cell-free apoptotic system and develop conditions that allow in vitro separation of the HMW and oligonucleosomal DNA fragmentation activities. In contrast to apoptosis associated with oligonucleosomal DNA fragmentation, the HMW DNA cleavage in apoptotic cells was accompanied by down-regulation of caspase-activated DNase (CAD) and was not affected by z-VAD-fmk, suggesting that the caspase/CAD pathway is not involved in the excision of DNA loop domains. We further demonstrate that nonapoptotic NB-2a cells contain a constitutively present nuclease activity located in the nuclear matrix fraction that possessed the properties of topoisomerase (topo) II and was capable of reproducing the pattern of HMW DNA cleavage that occurred in apoptotic cells. We demonstrate that the early stages of apoptosis induced by different stimuli were accompanied by activation of topo II-mediated HMW DNA cleavage that was reversible after removal of apoptotic inducers, and we present evidence of the involvement of topo II in the formation of HMW DNA fragments at the advanced stages of apoptosis. The results suggest that topo II is involved in caspase-independent excision of DNA loop domains during apoptosis, and this represents an alternative pathway of apoptotic DNA disintegration from CAD-driven caspase-dependent oligonucleosomal DNA cleavage.