Molecular cloning and expression of a cDNA encoding an apoptotic endonuclease DNase γ

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Abstract
An endonuclease named DNase γ has been purified from the nuclei of apoptotic rat thymocytes [Shiokawa, Ohyama, Yamada and Tanuma (1997) Biochem. J. 326, 675–681]. Here we report the molecular cloning of a cDNA encoding a 35 kDa precursor protein for rat DNase γ. A 1.6 kb mRNA coding for the DNase γ precursor is detected at high levels in spleen, lymph nodes, thymus and liver. By using reverse transcriptase-mediated PCR, expression of DNase γ mRNA is observed in kidney and testis but not in brain or heart. Analysis of recombinant DNase γ reveals that full-length DNase γ, including the N-terminal precursor, is an inactive proenzyme. The mature form of recombinant DNase γ, from which the N-terminal precursor has been removed, has the same properties as purified DNase γ: requirement for divalent cations, dependence on pH, sensitivity to Zn2+, and cleavage of chromosome DNA to nucleosomal units. In HeLa S3 cells stably transfected with the DNase γ cDNA, exogenously introduced DNase γ is activated by apoptotic stimuli; enhancement of DNA fragmentation, chromatin condensation and nuclear collapse are observed. These findings provide evidence for the involvement of DNase γ in DNA fragmentation and nuclear structural changes during apoptosis.

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