Role of DNAS1L3 in Ca2+- and Mg2+-dependent cleavage of DNA into oligonucleosomal and high molecular mass fragments

Ca2+- and Mg2+-dependent endonucleases have been implicated in DNA fragment ation during apoptosis. We have demonstrated that particular nucleases of t his type are inhibited by poly(ADP-ribosyl)ation and suggested that subsequ ent cleavage of PARP by caspase-3 might release these nucleases from poly(A DP-ribosyl)ation-induced inhibition. Hence, we purified and partially seque nced such a nuclease isolated from bovine seminal plasma and identified hum an, rat and mouse homologs of this enzyme. The extent of sequence homology among these nucleases indicates that these four proteins are orthologous me mbers of the family of DNase I-related enzymes. We demonstrate that the act ivation of the human homolog previously specified as DNAS1L3 can induce Ca2 +- and Mg2+-dependent DNA fragmentation in vitro and in vivo. RT-PCR analys is failed to detect DNAS1L3 mRNA in HeLa cells and nuclei isolated from the se cells did not exhibit internucleosomal DNA fragmentation when incubated in the presence of Ca2+ and Mg2+. However, nuclei isolated from HeLa cells that had been stably transfected with DNAS1L3 cDNA underwent such DNA fragm entation in the presence of both ions. The Ca2+ ionophore ionomycin also in duced internucleosomal DNA degradation in transfected but not in control He La cells. Transverse alternating field electrophoresis revealed that in nuc lei from transfected HeLa cells, but not in those from control cells, DNA w as cleaved into fragments of >1000 kb in the presence of Mg2+; addition of Ca2+ in the presence of Mg2+ resulted in processing of the >1000 kb fragmen ts into 50 kb and oligonucleosomal fragments. These results demonstrate tha t DNAS1L3 is necessary for Ca2+- and Mg2+-dependent cleavage of DNA into bo th oligonucleosomal and high molecular mass fragments in specific cell type s.