Cleavage preferences of the apoptotic endonuclease DFF40 (caspase- activated DNase or nuclease) on naked DNA and chromatin substrates

Piotr Widlak, Peng Li, Xiaodong Wang, William T. Garrard

Research output: Contribution to journalArticle

123 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)8226-8232
Number of pages7
JournalJournal of Biological Chemistry
Volume275
Issue number11
DOIs
StatePublished - Mar 17 2000

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Endonucleases
Chromatin
DNA Fragmentation
DNA
Micrococcal Nuclease
Substrates
Type II DNA Topoisomerase
Single-Stranded DNA
Deoxyribonuclease I
Enzymes
Cell Nucleus
HeLa Cells
rRNA Genes
Hydroxyl Radical
Histones
Ladders
Phosphates
caspase-activated deoxyribonuclease
RNA
Genes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Cleavage preferences of the apoptotic endonuclease DFF40 (caspase- activated DNase or nuclease) on naked DNA and chromatin substrates. / Widlak, Piotr; Li, Peng; Wang, Xiaodong; Garrard, William T.

In: Journal of Biological Chemistry, Vol. 275, No. 11, 17.03.2000, p. 8226-8232.

Research output: Contribution to journalArticle

Widlak, Piotr ; Li, Peng ; Wang, Xiaodong ; Garrard, William T. / Cleavage preferences of the apoptotic endonuclease DFF40 (caspase- activated DNase or nuclease) on naked DNA and chromatin substrates. In: Journal of Biological Chemistry. 2000 ; Vol. 275, No. 11. pp. 8226-8232.
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