We report here the reconstitution of a pathway that leads to the apoptotic changes in nuclei by using recombinant DNA fragmentation factor (DFF), a heterodimeric protein of 40 and 45 kDa. Coexpression of DFF40 and DFF45 is required to generate recombinant DFF, which becomes activated when DFF45 is cleaved by caspase-3. The cleaved fragments of DFF45 dissociate from the DFF40, the active component of DFF. Purified DFF40 exhibited an intrinsic DNase activity that was markedly stimulated by chromatin-associated proteins histone H1 and high mobility group proteins. DFF40 also triggered chromatin condensation when incubated with nuclei. These data suggest that DFF40 is sufficient to trigger both DNA fragmentation and chromatin condensation during apoptosis.

The re-discovery of exosomes as intercellular messengers with high potential for diagnostic and therapeutic utility has led to them becoming a popular topic of research in recent years. One of the essential research areas in this field is the characterization of exosomal cargo, which includes numerous non-randomly packed proteins and nucleic acids. Unexpectedly, a very challenging aspect of exploration of extracellular vesicles has turned out to be their effective and selective isolation. The plurality of developed protocols leads to qualitative and quantitative variability in terms of the obtained exosomes, which significantly affects the results of downstream analyses and makes them difficult to compare, reproduce and interpret between research groups. Currently, there is a general consensus among the exosome-oriented community concerning the urgent need for the optimization and standardization of methods employed for the purification of these vesicles. Hence, we review here several strategies for exosome preparation including ultracentrifugation, chemical precipitation, affinity capturing and filtration techniques. The advantages and disadvantages of different approaches are discussed with special emphasis being placed on their adequacy for proteomics applications, which are particularly sensitive to sample quality. We conclude that certain methods, exemplified by ultracentrifugation combined with iodixanol density gradient centrifugation or gel filtration, although labor-intensive, provide superior quality exosome preparations suitable for reliable analysis by mass spectrometry.

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.

DNA fragmentation factor (DFF) is a heterodimeric protein composed of 45-kDa (DFF45) and 40-kDa (DFF40) subunits, a protein that mediates regulated DNA fragmentation and chromatin condensation in response to apoptotic signals. DFF45 is a specific molecular chaperone and an inhibitor for the nuclease activity of DFF40. Previous studies have shown that upon cleavage of DFF45 by caspase-3, the nuclease activity of DFF40 is relieved of inhibition. Here we further investigate the mechanism of DFF40 activation. We demonstrate that DFF45 can also be cleaved and inactivated by caspase-7 but not by caspase-6 and caspase-8. The cleaved DFF45 fragments dissociate from DFF40, allowing DFF40 to oligomerize to form a large functional complex that cleaves DNA by introducing double strand breaks. Histone H1 directly interacts with DFF, confers DNA binding ability to DFF, and stimulates the nuclease activity of DFF40 by increasing its K _cat and decreasing itsK _m.

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 ≥ 50 kb in length, followed by inter- and intranucleosomal DNA cleavages with products possessing significant internal single-stranded nicks spaced at nucleosomal (∼190 bases) and subnucleosomal (∼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.