Single-strand breaks in agarose-embedded chromatin of nonapoptotic cells

Loop-size chromatin fragmentation frequently observed upon apoptotic cell d eath is thought to be initiated by ss nicks. Here we show that the agarose- embedded, deproteinized chromatin of normal, nonapoptotic murine and human cells, as well as yeast protoplasts, falls apart to 50-300 kb ss fragments upon heat denaturation, as revealed by urea-TAE field-inversion agarose gel electrophoresis resolving ss and ds fragments alike. These data were in li ne with S1digestion experiments. The nicks (gaps) observed are best explain ed either by enzymatic cleavages occurring upon cell lysis instantaneously or by preexisting discontinuities becoming manifest upon heat denaturation. These discontinuities go unnoticed in the usual nondenaturaing circumstanc es but seem to be inevitably present in any DNA preparation, The loop-size ds DNA fragmentation in apoptosis may be based on these pre-existing or "re ady-to-go" (upon cell lysis) ss discontinuities of the normal cellular chro matin. (C) 1999 Academic Press.