Chromatin collapse during caspase-dependent apoptotic cell death requires DNA fragmentation factor, 40-kDa subunit-/caspase-activated deoxyribonuclease- mediated 3′-OH single-strand DNA breaks

Victoria Iglesias-Guimarais, Estel Gil-Guiñon, María Sánchez-Osuna, Elisenda Casanelles, Mercè García-Belinchón, Joan X. Comella, Victor J. Yuste

Research output: Contribution to journalArticleResearchpeer-review

22 Citations (Scopus)

Abstract

Apoptotic nuclear morphology and oligonucleosomal double-strand DNA fragments (also known as DNA ladder) are considered the hallmarks of apoptotic cell death. From a classic point of view, these two processes occur concomitantly. Once activated, DNA fragmentation factor, 40-kDa subunit (DFF40)/caspase-activated DNase (CAD) endonuclease hydrolyzes the DNA into oligonucleosomal-size pieces, facilitating the chromatin package. However, the dogma that the apoptotic nuclear morphology depends on DNA fragmentation has been questioned. Here, we use different cellular models, including MEF CAD -/- cells, to unravel the mechanism by which DFF40/CAD influences chromatin condensation and nuclear collapse during apoptosis. Upon apoptotic insult, SK-N-AS cells display caspase-dependent apoptotic nuclear alterations in the absence of internucleosomal DNA degradation. The overexpression of a wild-type form of DFF40/CAD endonuclease, but not of different catalytic-null mutants, restores the cellular ability to degrade the chromatin into oligonucleosomal-length fragments. We show that apoptotic nuclear collapse requires a 3′-OH endonucleolytic activity even though the internucleosomal DNA degradation is impaired. Moreover, alkaline unwinding electrophoresis and In Situ End-Labeling (ISEL)/In Situ Nick Translation (ISNT) assays reveal that the apoptotic DNA damage observed in the DNA ladder-deficient SK-N-AS cells is characterized by the presence of single-strand nicks/breaks. Apoptotic single-strand breaks can be impaired by DFF40/CAD knockdown, abrogating nuclear collapse and disassembly. In conclusion, the highest order of chromatin compaction observed in the later steps of caspase-dependent apoptosis relies on DFF40/CAD-mediated DNA damage by generating 3′-OH ends in single-strand rather than doublestrand DNA nicks/breaks. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.
Original languageEnglish
Pages (from-to)9200-9215
JournalJournal of Biological Chemistry
Volume288
DOIs
Publication statusPublished - 29 Mar 2013

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