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| Home > Articles > Published articles > 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 |
| Home > Articles > Published articles > 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 |
(Hospital Universitari Vall d'Hebron) (Universitat Autònoma de Barcelona. Departament de Bioquímica i de Biologia Molecular)
| Date: | 2013 |
| 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, DFF40/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, the alkaline unwinding electrophoresis and the ISEL/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 (SSBs). Apoptotic SSBs 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. |
| Grants: | Ministerio de Ciencia e Innovación SAF2011-24081 Ministerio de Economía y Competitividad SAF2012-31485 Ministerio de Ciencia e Innovación SAF2010-19953 Ministerio de Sanidad y Consumo CB06/05/0042 Agència de Gestió d'Ajuts Universitaris i de Recerca 2009/SGR-346 Ministerio de Ciencia e Innovación TRA2009-0185 Ministerio de Ciencia e Innovación BES-2099-028572 |
| Note: | Altres ajuts: MEC programa Ramón y Cajal |
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| Language: | Anglès |
| Document: | Article ; recerca ; Versió publicada |
| Subject: | Apoptosis ; Caspase ; DNA damage ; DNase ; Neuroblastoma ; DFF40/CAD ; Apoptotic nuclear morphology ; Oligonucleosomal DNA degradation ; Single-strand DNA nicks/breaks |
| Published in: | Journal of biological chemistry, Vol. 288, Num. 13 (Mar 2013) , p. 9200-9215, ISSN 1083-351X |
