Apoptotic nuclease assays.
One of the defining biochemical characteristics of apoptosis is the degradation of chromatin into regularly sized (oligonucleosomal and approximately 30- to 50-kb) fragments. Because destruction of the genome represents a clear commitment to death, considerable interest has focused on this component of apoptosis and numerous assays have been developed to assess the relevant nucleases involved. These assays fall into two major categories: (1) those independent of chromatin structure and (2) those dependent on chromatin structure. The chromatin-independent assays (plasmid degradation assay and radioactive gel assay) examine the ability to degrade naked DNA and are advantageous because of their simplicity and speed and ability to analyze single nucleases or mixtures of nucleases. However, these assays do not mimic the conditions present in normal cells and consequently do not assess the ability of an enzyme to function in apoptosis. In contrast, chromatin structure-dependent assays (nuclear autodigestion and HeLa nuclei assay) present intact chromatin to either endogenous or exogenous enzymes and assess the ability to degrade chromatin in a manner that recapitulates the genomic destruction seen in vivo. Detailed protocols are discussed for both classes of assays. These assays have been instrumental in the identification of several apoptotic nucleases.
University of North Carolina at Charlotte 28223, USA.
F M
FM
Hughes
J A
JA
Cidlowski
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002460:Cell Line; D002467:Cell Nucleus / Q000502:physiology / Q000648:ultrastructure*; D002843:Chromatin / Q000502:physiology* / Q000648:ultrastructure; D004247:DNA / Q000032:analysis* / Q000096:biosynthesis; D053938:DNA Fragmentation; D003851:Deoxyribonucleases / Q000032:analysis*; D003856:Deoxyuracil Nucleotides / Q000378:metabolism; D003907:Dexamethasone / Q000494:pharmacology; D004587:Electrophoresis, Agar Gel / Q000379:methods; D006367:HeLa Cells; D006801:Humans; D007202:Indicators and Reagents; D010761:Phosphorus Radioisotopes; D010957:Plasmids / Q000378:metabolism; D013601:T-Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000502:physiology
D002843:Chromatin; D003856:Deoxyuracil Nucleotides; D007202:Indicators and Reagents; D010761:Phosphorus Radioisotopes; C027078:deoxyuridine triphosphate; D003907:Dexamethasone; D004247:DNA; D003851:Deoxyribonucleases