Nucleic Acids Research Advance Access originally published online on July 10, 2007
Nucleic Acids Research 2007 35(13):e93; doi:10.1093/nar/gkm486
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Nucleic Acids Research, 2007, Vol. 35, No. 13 e93
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Methods Online |
Engineered apoptotic nucleases for chromatin research
1Department of Molecular Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390, USA and 2Faculty of Biotechnology, University of Rzeszow, 36-100 Werynia, Poland
*To whom correspondence should be addressed. Tel: +1 214 648 1924; Fax: +1 214 648 1915; Email: william.garrard{at}utsouthwestern.edu
Received April 18, 2007. Revised May 28, 2007. Accepted June 5, 2007.
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.