Chromosomal DNA degradation is critical for cell death execution and is a hallmark of apoptosis, yet little is known about how this process is executed. Classical genetic screens have been ineffective at identifying the components of the DNA degradation machinery and mutant alleles in known apoptotic nucleases do not display easily detectable phenotypes. In order to overcome this problem, we have used the TUNEL assay to conduct an RNAi-based functional genomic screen to identify additional nucleases involved in apoptotic DNA degradation. From an initial screen of 77 open reading frames (ORFs) encoding C. elegans nucleases, cyclophilins and topoisomerases, we have isolated nine cell death-related nucleases (crn genes), two of which (
cps-6 and
nuc-1) were previously known to function in apoptotic DNA degradation. Two genes,
crn-6 and
cyp-13, encode homologues of mammalian genes previously implicated in apoptotic DNA degradation (DNase II and cyclophilin family proteins, respectively), demonstrating that these genes likely play a conserved role in DNA degradation. Genetic and phenotypic analyses suggest that these crn genes likely function in two distinct, partially independent pathways to mediate apoptotic DNA degradation, with
cps-6,
crn-1,
crn-4,
crn-5, and
cyp-13 functioning in one pathway and
crn-2 and
crn-3 acting in the other pathway. Interestingly, five of the nucleases (including CPS-6) that act in the same pathway and at least one non-nuclease component, WAH-1 (worm apoptosis-inducing factor homologue), appear to interact with one another in vitro and may assemble into a protein complex to mediate apoptotic DNA degradation in vivo. We have named this complex the degradeosome. Currently, we are expanding the screen to include recently annotated nucleases and nuclease related genes to identify the nuclease(s) functioning upstream of
cps-6 to initiate apoptotic DNA degradation and to identify additional components of the degradeosome. Furthermore, we are conducting a genetic suppressor screen to isolate mutations that suppress
crn-2/3 or
cps-6-induced ectopic cell killing to identify additional nuclease and non-nuclease components that participate in two different DNA degradation pathways. Finally, we are characterizing several cps (
ced-3 protease suppressor) mutants that are also defective in apoptotic DNA degradation. Taken together, these studies should advance our understanding of the molecular mechanisms of apoptotic DNA degradation.