Abstract
Endonuclease G (endoG) is released from mitochondria during apoptosis and is in part responsible for internucleosomal DNA cleavage. Here we report the action of the purified human recombinant form of this endonuclease on naked DNA and chromatin substrates. The addition of the protein to isolated nuclei from non-apoptotic cells first induces higher order chromatin cleavage into DNA fragments > or = 50 kb in length, followed by inter- and intranucleosomal DNA cleavages with products possessing significant internal single-stranded nicks spaced at nucleosomal ( approximately 190 bases) and subnucleosomal ( approximately 10 bases) periodicities. We demonstrate that both exonucleases and DNase I stimulate the ability of endoG to generate double-stranded DNA cleavage products at physiological ionic strengths, suggesting that these activities work in concert with endoG in apoptotic cells to ensure efficient DNA breakdown.
Highlights
Endonuclease G is released from mitochondria during apoptosis and is in part responsible for internucleosomal DNA cleavage
The addition of the protein to isolated nuclei from nonapoptotic cells first induces higher order chromatin cleavage into DNA fragments > 50 kb in length, followed by inter- and intranucleosomal DNA cleavages with products possessing significant internal single-stranded nicks spaced at nucleosomal (ϳ190 bases) and subnucleosomal (ϳ10 bases) periodicities. We demonstrate that both exonucleases and DNase I stimulate the ability of Endonuclease G (endoG) to generate double-stranded DNA cleavage products at physiological ionic strengths, suggesting that these activities work in concert with endoG in apoptotic cells to ensure efficient DNA breakdown
EndoG is released from the intermembrane space of mitochondria during apoptosis in a caspase-independent fashion and represents a novel pathway for nuclear DNA breakdown [9, 10]
Summary
The addition of the protein to isolated nuclei from nonapoptotic cells first induces higher order chromatin cleavage into DNA fragments > 50 kb in length, followed by inter- and intranucleosomal DNA cleavages with products possessing significant internal single-stranded nicks spaced at nucleosomal (ϳ190 bases) and subnucleosomal (ϳ10 bases) periodicities. We demonstrate that both exonucleases and DNase I stimulate the ability of endoG to generate double-stranded DNA cleavage products at physiological ionic strengths, suggesting that these activities work in concert with endoG in apoptotic cells to ensure efficient DNA breakdown. Both cytochrome c and SMAC/DIABLO are involved in caspase activation, whereas AIF and endoG have been associated with one of the hallmarks of the terminal stages of apoptosis, DNA breakdown [11, 12]
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