Ionic and cofactor requirements for the activity of the apoptotic endonuclease DFF40/CAD

P. Widlak, W. T. Garrard

Research output: Contribution to journalArticle

40 Citations (Scopus)

Abstract

The endonuclease DFF40/CAD mediates regulated DNA fragmentation and chromatin condensation in cells undergoing apoptosis. Here we report the enzyme's co-factor requirements, and demonstrate that the ionic changes that occur in apoptotic cells maximize DFF40/CAD activity. The nuclease requires Mg2+, exhibits a trace of activity in the presence of Mn2+, is not costimulated by Ca2+, is inhibited by Zn2+ or Cu2+, and has high activity over a rather broad pH range (7.0-8.5). The enzyme is thermally unstable, and is rapidly inactivated at 42°C. Enzyme activity is markedly affected by ionic strength. At the optimal [K+] of 50-125 mM, which is in the range of the cytoplasmic [K+] for cells undergoing apoptosis, the activity of DFF40/CAD for naked DNA cleavage is about 100-fold higher than at 0 or 200 mM [K+]. Although these ranges of ionic strength do not affect DFF40 homo-oligomer formation, at higher ionic strengths the enzyme introduces single-stranded nicks into supercoiled DNA.

Original languageEnglish (US)
Pages (from-to)125-130
Number of pages6
JournalMolecular and Cellular Biochemistry
Volume218
Issue number1-2
DOIs
StatePublished - 2001

Fingerprint

Endonucleases
Ionic strength
Computer aided design
Osmolar Concentration
Enzymes
Apoptosis
Superhelical DNA
DNA
Enzyme activity
Oligomers
DNA Cleavage
Chromatin
Condensation
DNA Fragmentation
Cells

Keywords

  • Apoptosis
  • CAD
  • Chromatin
  • Co-factor requirements
  • DFF
  • Nuclease

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Molecular Biology
  • Genetics
  • Cell Biology

Cite this

Ionic and cofactor requirements for the activity of the apoptotic endonuclease DFF40/CAD. / Widlak, P.; Garrard, W. T.

In: Molecular and Cellular Biochemistry, Vol. 218, No. 1-2, 2001, p. 125-130.

Research output: Contribution to journalArticle

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abstract = "The endonuclease DFF40/CAD mediates regulated DNA fragmentation and chromatin condensation in cells undergoing apoptosis. Here we report the enzyme's co-factor requirements, and demonstrate that the ionic changes that occur in apoptotic cells maximize DFF40/CAD activity. The nuclease requires Mg2+, exhibits a trace of activity in the presence of Mn2+, is not costimulated by Ca2+, is inhibited by Zn2+ or Cu2+, and has high activity over a rather broad pH range (7.0-8.5). The enzyme is thermally unstable, and is rapidly inactivated at 42°C. Enzyme activity is markedly affected by ionic strength. At the optimal [K+] of 50-125 mM, which is in the range of the cytoplasmic [K+] for cells undergoing apoptosis, the activity of DFF40/CAD for naked DNA cleavage is about 100-fold higher than at 0 or 200 mM [K+]. Although these ranges of ionic strength do not affect DFF40 homo-oligomer formation, at higher ionic strengths the enzyme introduces single-stranded nicks into supercoiled DNA.",
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AB - The endonuclease DFF40/CAD mediates regulated DNA fragmentation and chromatin condensation in cells undergoing apoptosis. Here we report the enzyme's co-factor requirements, and demonstrate that the ionic changes that occur in apoptotic cells maximize DFF40/CAD activity. The nuclease requires Mg2+, exhibits a trace of activity in the presence of Mn2+, is not costimulated by Ca2+, is inhibited by Zn2+ or Cu2+, and has high activity over a rather broad pH range (7.0-8.5). The enzyme is thermally unstable, and is rapidly inactivated at 42°C. Enzyme activity is markedly affected by ionic strength. At the optimal [K+] of 50-125 mM, which is in the range of the cytoplasmic [K+] for cells undergoing apoptosis, the activity of DFF40/CAD for naked DNA cleavage is about 100-fold higher than at 0 or 200 mM [K+]. Although these ranges of ionic strength do not affect DFF40 homo-oligomer formation, at higher ionic strengths the enzyme introduces single-stranded nicks into supercoiled DNA.

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