title: Unique features of the apoptotic endonuclease DFF40/CAD relative to micrococcal nuclease as a structural probe for chromatin.
abstract: The gold standard for studies of nucleosomal chromatin structure for the past 30 years has been the enzyme micrococcal nuclease (MNase). During the course of our studies on the elucidation of the mechanism of action of the apoptotic nuclease DNA fragmentation factor-40 / caspase-activated deoxyribonuclease (DFF40/CAD) on naked DNA and chromatin substrates, it became clear that this enzyme is superior in certain respects to MNase for studying several aspects of chromatin structure. Here we review our published results supporting this statement. Relative to MNase, we have found that DFF40/CAD has the following properties: (i) it does not cut within nucleosomes to generate subnucleosomal DNA fragments; (ii) it is more specific for the linker regions between nucleosomes; (iii) it lacks exonuclease activity; (iv) it is specific for double-stranded DNA and makes exclusively double-stranded breaks; and (v) it attacks histone-H1-containing chromatin more efficiently. Taken together, these facts explain why DFF40/CAD generates sharper oligonucleosomal DNA ladders compared with those generated by MNase. We therefore recommend the following uses for DFF40/CAD for chromatin research: nucleosome isolation, chromatin-remodeling assays, repeat length measurements, and nucleosome-positioning assays along specific sequences. Other uses include footprinting assays of transcription factor positions, shearing chromatin for immunopreciptitation experiments (ChIP), and shearing DNA for recombinant DNA library preparation or for shotgun cloning for sequencing.
authors.affiliation: Department of Molecular Biology, University of TX Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390-9148, USA.
authors.forename: Piotr
authors.initials: P
authors.lastname: Widlak
authors.forename: William T
authors.initials: WT
authors.lastname: Garrard
mesh_terms: D017209:Apoptosis; D001483:Base Sequence; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism; D002843:Chromatin / Q000235:genetics* / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D053837:DNA Cleavage; D003851:Deoxyribonucleases / Q000235:genetics* / Q000378:metabolism; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006657:Histones / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008836:Micrococcal Nuclease / Q000235:genetics* / Q000378:metabolism; D015335:Molecular Probes / Q000235:genetics / Q000378:metabolism; D009707:Nucleosomes / Q000235:genetics / Q000378:metabolism; D000075223:Poly-ADP-Ribose Binding Proteins
chemical_list: D002843:Chromatin; D006657:Histones; D015335:Molecular Probes; D009707:Nucleosomes; D000075223:Poly-ADP-Ribose Binding Proteins; D004247:DNA; C113696:DFFB protein, human; D003851:Deoxyribonucleases; D004720:Endonucleases; C109663:caspase-activated deoxyribonuclease; D008836:Micrococcal Nuclease; D053148:Caspase 3