DNA FRAGMENTATION DURING APOPTOSIS IS CAUSED BY FREQUENT SINGLE-STRAND CUTS

One of the hallmarks of apoptosis is the digestion of genomic DNA by a n endonuclease, generating a ladder of small fragments of double-stran ded DNA. We have examined the nature of the DNA breaks produced in mou se thymocytes triggered to undergo apoptosis by steroids or by stimula tion of the T cell receptor. Whereas the typical ladder pattern of oli gonucleosomal fragments was observed after agarose gel electrophoresis , numerous single-strand cuts were detected after electrophoresis unde r denaturing conditions. Single-strand nicks were found to be very fre quent in the internucleosomal regions, but also to occur in the core p article-associated DNA. An identical pattern of single-strand nicks wa s obtained when chromatin DNA was exposed to the single-strand cleavin g deoxyribonuclease I. The nicked DNA fragments, extracted from apopto tic thymocytes, were sensitive to the action of S1-nuclease. We propos e that DNA fragmentation induced during apoptosis is not due to a doub le-strand cutting enzyme as previously postulated, but rather is the r esult of single-strand breaks. This ensures the dissociation of the DN A molecule at sites where cuts are found within close proximity.