The major apoptotic nuclease, DNA fragmentation factor (DFF), also termed Caspase-activated DNase (CAD), is primarily responsible for internucleosomal DNA cleavage during the terminal stages of apoptosis. In non-apoptotic cells, DFF exists in the nucleus as a heterodimer, composed of a 45 kD chaperone and inhibitor subunit (DFF45/ICAD) and a 40 kD latent nuclease subunit DFF40/CAD. Activation of the nuclease depends on caspase-3-mediated cleavage of DFF45/ICAD inhibitor and formation of DFF40/CAD homooligomers. Caspase-activated DFF40/CAD homo-oligomers can further interact with additional activators or inhibitors; few of them had been identified so far.

In aim to identify novel proteins that interact with DFF40/CAD we have used the yeast-two hybrid system. Yeast S. cerevisiae strain AH105 was transformed with human DFF40 (cloned into pGBT9 vector) and then mated with S. cerevisiae strain Y187 carrying mouse brain embryo cDNA library. Several hybrid clones have been identified. Majority of such clones carried cDNA of DFF45/ICAD, major partner of DFF40/CAD nuclease. However, a few clones contained cDNA encoding proteins never reported to interact with the apoptotic nuclease, including: Annexin A2, Rab 10, Ankyrin 2, Heme-regulated initiation factor 2 alpha kinase (Eif2ak1). These proteins are under further investigation.

This work was supported by the Ministry of Science, Grant N301 058 31/1763.

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