title: Engineered apoptotic nucleases for chromatin research.
abstract: We have created new genomics tools for chromatin research by genetically engineering the human and mouse major apoptotic nucleases that are responsible for internucleosomal DNA cleavage, DNA fragmentation factor (DFF). Normally, in its inactive form, DFF is a heterodimer composed of a 45-kDa chaperone inhibitor subunit (DFF45 or ICAD), and a 40-kDa latent endonuclease subunit (DFF40 or CAD). Upon caspase-3 cleavage of DFF45, DFF40 forms active endonuclease homo-oligomers. Although Saccharomyces cerevisiae lacks DFF, expression of caspase-3 is lethal in this organism, but expression of the highly sequence-specific tobacco etch virus protease (TEVP) is harmless. Therefore, we inserted TEVP cleavage sites immediately downstream of the two caspase-3 cleavage sites within DFF45, generating a novel form of DFF (DFF-T) whose nuclease activity proved to be exclusively under the control of TEVP. We demonstrate that co-expression of TEVP and DFF-T under galactose control results in nucleosomal DNA laddering and cell death in S. cerevisiae. We also created synthetic DFF genes with optimized codons for high-level expression in Eschericia coli or S. cerevisiae. We further demonstrate the excellence of the synthetic gene products for in vitro mapping of the nucleosome positions and hypersensitive sites in specific genes such as the yeast PHO5.
authors.affiliation: Department of Molecular Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390, USA.
authors.forename: Fei
authors.initials: F
authors.lastname: Xiao
authors.forename: Piotr
authors.initials: P
authors.lastname: Widlak
authors.forename: William T
authors.initials: WT
authors.lastname: Garrard
mesh_terms: D000135:Acid Phosphatase; D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000235:genetics* / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D018983:DNA Footprinting; D003851:Deoxyribonucleases / Q000235:genetics* / Q000378:metabolism; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005813:Genes, Synthetic; D023281:Genomics; D006801:Humans; D007202:Indicators and Reagents; D051379:Mice; D016296:Mutagenesis; D009707:Nucleosomes / Q000737:chemistry*; D010957:Plasmids / Q000378:metabolism; D000075223:Poly-ADP-Ribose Binding Proteins; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000032:analysis; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics
chemical_list: D051017:Apoptosis Regulatory Proteins; D007202:Indicators and Reagents; D009707:Nucleosomes; D000075223:Poly-ADP-Ribose Binding Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C109664:caspase-activated DNase inhibitor; D004247:DNA; D003851:Deoxyribonucleases; C499445:Dffb protein, mouse; D000135:Acid Phosphatase; C488916:PHO5 protein, S cerevisiae; D010450:Endopeptidases; C099488:TEV protease