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J Biol Chem, Vol. 275, Issue 11, 8226-8232, March 17, 2000

Cleavage Preferences of the Apoptotic Endonuclease DFF40 (Caspase-activated DNase or Nuclease) on Naked DNA and Chromatin Substrates^*

Piotr Widlak^§, Peng Li^¶, Xiaodong Wang^**, and William T. Garrard

From the Departments of  Molecular Biology and  Biochemistry and the ^** Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas 75235

Here we report the co-factor requirements for DNA fragmentation factor (DFF) endonuclease and characterize its cleavage sites on naked DNA and chromatin substrates. The endonuclease exhibits a pH optimum of 7.5, requires Mg²⁺, not Ca²⁺, and is inhibited by Zn²⁺. The enzyme generates blunt ends or ends with 1-base 5'-overhangs possessing 5'-phosphate and 3'-hydroxyl groups and is specific for double- and not single-stranded DNA or RNA. DFF endonuclease has a moderately greater sequence preference than micrococcal nuclease or DNase I, and the sites attacked possess a dyad axis of symmetry with respect to purine and pyrimidine content. Using HeLa cell nuclei or chromatin reconstituted on a 5 S rRNA gene tandem array, we prove that the enzyme attacks chromatin in the internucleosomal linker, generating oligonucleosomal DNA ladders sharper than those created by micrococcal nuclease. Histone H1, high mobility group-1, and topoisomerase II activate DFF endonuclease activity on naked DNA substrates but much less so on chromatin substrates. We conclude that DFF is a useful reagent for chromatin research.

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^* This work was supported in part by the Polish State Committee for Scientific Research Grant 6P04A01317 (to P. W.), American Cancer Society Grant RE258, National Institutes of Health Grant GMRO1-55942, Robert A. Welch Foundation Grant I-1412 (to X. W.), National Institutes of Health Grant GMRO1-29935, and Robert A. Welch Foundation Grant I-0823 (to W. T. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Present address: Dept. of Experimental and Clinical Radiobiology, Center of Oncology, 44-100 Gliwice, Poland.

^¶ Present address: Inst. of Molecular and Cell Biology, 30 Medical Dr., Singapore 117609.

To whom correspondence and reprint requests should be addressed: Dept. of Molecular Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9148. Tel.: 214-648-1924; Fax: 214-648-1909; E-mail: garrard@utsw.swmed.edu.

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Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

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