Unique features of the apoptotic endonuclease DFF40/CAD relative to micrococcal nuclease as a structural probe for
Piotr Widlak1, William T Garrard
1Department of Molecular Biology, University of TX Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390-9148, USA.
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.
Insights
DNA fragmentation factor-40 / caspase-activated deoxyribonuclease (DFF40/CAD) offers superior chromatin structure analysis compared to micrococcal nuclease (MNase). DFF40/CAD provides sharper DNA ladders and is recommended for various chromatin research applications.
Area of Science:
- Molecular Biology
- Chromatin Biology
- Enzymology
Context:
- Micrococcal nuclease (MNase) has been the standard for chromatin structure studies for decades.
- Investigating the apoptotic nuclease DNA fragmentation factor-40 / caspase-activated deoxyribonuclease (DFF40/CAD) revealed its advantages over MNase.
- Published results demonstrate DFF40/CAD's superior properties for chromatin analysis.
Purpose:
- To review published results highlighting the advantages of DFF40/CAD over MNase in chromatin structure studies.
- To recommend specific applications of DFF40/CAD in chromatin research.
Summary:
- DFF40/CAD exhibits unique properties compared to MNase: it avoids cutting within nucleosomes, specifically targets linker DNA, lacks exonuclease activity, creates only double-stranded DNA breaks, and efficiently processes histone-H1-containing chromatin.
- These characteristics result in sharper oligonucleosomal DNA ladders generated by DFF40/CAD than by MNase.
Impact:
- DFF40/CAD is recommended for nucleosome isolation, chromatin remodeling assays, repeat length measurements, and nucleosome positioning assays.
- Further applications include transcription factor footprinting, chromatin shearing for ChIP, and DNA shearing for library preparation and sequencing.
