title: 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.
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 double-strand DNA nicks/breaks.
authors: Cell Death, Senescence, and Survival Group, Departament de Bioquímica i Biologia Molecular and Institut de Neurociències, Facultat de Medicina, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain.
authors: Victoria
authors: V
authors: Iglesias-Guimarais
authors: Estel
authors: E
authors: Gil-Guiñon
authors: María
authors: M
authors: Sánchez-Osuna
authors: Elisenda
authors: E
authors: Casanelles
authors: Mercè
authors: M
authors: García-Belinchón
authors: Joan X
authors: JX
authors: Comella
authors: Victor J
authors: VJ
authors: Yuste
mesh_terms: D000818:Animals; D017209:Apoptosis; D006690:Bisbenzimidazole / Q000494:pharmacology; D020169:Caspases / Q000378:metabolism*; D016923:Cell Death; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002843:Chromatin / Q000737:chemistry* / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D053904:DNA Breaks, Single-Stranded; D004249:DNA Damage; D053938:DNA Fragmentation; D003851:Deoxyribonucleases / Q000378:metabolism*; D004720:Endonucleases / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D051379:Mice; D008954:Models, Biological; D009154:Mutation; D009447:Neuroblastoma / Q000378:metabolism; D009707:Nucleosomes / Q000378:metabolism; D000075223:Poly-ADP-Ribose Binding Proteins; D014343:Trypan Blue / Q000494:pharmacology
chemical_list: D002843:Chromatin; D009707:Nucleosomes; D000075223:Poly-ADP-Ribose Binding Proteins; D004247:DNA; C113696:DFFB protein, human; D003851:Deoxyribonucleases; C499445:Dffb protein, mouse; D004720:Endonucleases; D020169:Caspases; D014343:Trypan Blue; D006690:Bisbenzimidazole