Single-strand breaks in agarose-embedded chromatin of nonapoptotic cells.
Loop-size chromatin fragmentation frequently observed upon apoptotic cell death is thought to be initiated by ss nicks. Here we show that the agarose-embedded, deproteinized chromatin of normal, non-apoptotic 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 line with S1digestion experiments. The nicks (gaps) observed are best explained 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 circumstances 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 "ready-to-go" (upon cell lysis) ss discontinuities of the normal cellular chromatin.
Varga
T
T
Department of Biophysics and Cell Biology, University Medical School of Debrecen, 4012 Debrecen, Nagyerdei krt. 98, Hungary.
Szilágyi
I
I
Szabó
G
G
D000818:Animals; D002460:Cell Line; D002843:Chromatin / Q000737:chemistry* / Q000648:ultrastructure; D004249:DNA Damage; D053938:DNA Fragmentation; D004277:DNA, Single-Stranded / Q000737:chemistry; D004587:Electrophoresis, Agar Gel; D016521:Electrophoresis, Gel, Pulsed-Field; D006358:Hot Temperature; D006801:Humans; D051379:Mice; D009691:Nucleic Acid Denaturation; D012441:Saccharomyces cerevisiae; D012685:Sepharose; D015719:Single-Strand Specific DNA and RNA Endonucleases; D014508:Urea
D002843:Chromatin; D004277:DNA, Single-Stranded; D014508:Urea; D012685:Sepharose; D015719:Single-Strand Specific DNA and RNA Endonucleases