Molecular cloning and expression of a cDNA encoding an apoptotic endonuclease DNase gamma.
An endonuclease named DNase gamma 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 gamma. A 1.6 kb mRNA coding for the DNase gamma precursor is detected at high levels in spleen, lymph nodes, thymus and liver. By using reverse transcriptase-mediated PCR, expression of DNase gamma mRNA is observed in kidney and testis but not in brain or heart. Analysis of recombinant DNase gamma reveals that full-length DNase gamma, including the N-terminal precursor, is an inactive proenzyme. The mature form of recombinant DNase gamma, from which the N-terminal precursor has been removed, has the same properties as purified DNase gamma: 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 gamma cDNA, exogenously introduced DNase gamma 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 gamma in DNA fragmentation and nuclear structural changes during apoptosis.
Shiokawa
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Department of Biochemistry, Faculty of Pharmaceutical Sciences, Science University of Tokyo, Shinjuku-ku, Tokyo 162, Japan.
Tanuma
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S
D000818:Animals; D017209:Apoptosis / Q000235:genetics* / Q000502:physiology; D001483:Base Sequence; D002843:Chromatin / Q000378:metabolism; D003001:Cloning, Molecular; D053938:DNA Fragmentation; D018076:DNA, Complementary / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004789:Enzyme Activation; D005982:Glutathione Transferase / Q000096:biosynthesis; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009928:Organ Specificity; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D002843:Chromatin; D018076:DNA, Complementary; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D004706:Endodeoxyribonucleases; C105124:deoxyribonuclease gamma