Abstract
DNA fragmentation factor (DFF) is a complex of the DNase DFF40 (CAD) and its chaperone/inhibitor DFF45 (ICAD-L) that can be activated during apoptosis to induce DNA fragmentation. Here, we demonstrate that DFF directly binds to DNA in vitro without promoting DNA cleavage. DNA binding by DFF is mediated by the nuclease subunit, which can also form stable DNA complexes after release from DFF. Recombinant and reconstituted DFF is catalytically inactive yet proficient in DNA binding, demonstrating that the nuclease subunit in DFF is inhibited in DNA cleavage but not in DNA binding, revealing an unprecedented mode of nuclease inhibition. Activation of DFF in the presence of naked DNA or isolated nuclei stimulates DNA degradation by released DFF40 (CAD). In transfected HeLa cells transiently expressed DFF associates with chromatin, suggesting that DFF could be activated during apoptosis in a DNA-bound state.
Highlights
DNA fragmentation is a biochemical hallmark of apoptotic cell death that can be achieved by the action of several nucleases involved in various apoptotic signal transduction pathways [1,2,3,4]
DNA binding by DNA fragmentation factor (DFF) prior to or concomitant with its activation by caspase-3 stimulates the activity of the released nuclease DFF40 (CAD) on naked DNA and isolated nuclei, suggesting that during apoptosis nuclear DFF can be activated in a DNAbound state
More than 95% of DFF45 was detectable in the Triton X-100 soluble fraction, whereas DFF was split among the Triton X-100 soluble fraction and the chromatin fraction (Fig. 6G). These results strongly suggest that the nuclease/inhibitor complex DFF, but not the inhibitory subunit DFF45 (ICAD-L) alone, is able to associate with chromatin corroborating the observations made on DNA binding by DFF in vitro
Summary
DNA fragmentation is a biochemical hallmark of apoptotic cell death that can be achieved by the action of several nucleases involved in various apoptotic signal transduction pathways [1,2,3,4]. DFF40 (CAD) directly binds to histone H1, suggesting that this protein perhaps targets the nuclease to the DNA linker region where nucleosomal DNA fragmentation occurs (6, 19 –21). We provide evidence that DFF in addition to the above mentioned interactions can directly interact with DNA in vitro without inducing DNA cleavage and is able to associate with chromatin in transfected HeLa cells. This is the first known case of a DNase-inhibitor complex in which DNA binding occurs but DNA cleavage is blocked. DNA binding by DFF prior to or concomitant with its activation by caspase-3 stimulates the activity of the released nuclease DFF40 (CAD) on naked DNA and isolated nuclei, suggesting that during apoptosis nuclear DFF can be activated in a DNAbound state
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