Yakovlev A G, Wang G, Stoica B A, Simbulan-Rosenthal C M, Yoshihara K, Smulson M E
Department of Biochemistry and Molecular Biology, Georgetown University School of Medicine, Basic Science Building, Room 351, 3900 Reservoir Road NW, Washington, DC 20007, USA.
Nucleic Acids Res. 1999 May 1;27(9):1999-2005. doi: 10.1093/nar/27.9.1999.
Ca2+- and Mg2+-dependent endonucleases have been implicated in DNA fragmentation during apoptosis. We have demonstrated that particular nucleases of this type are inhibited by poly(ADP-ribosyl)ation and suggested that subsequent cleavage of PARP by caspase-3 might release these nucleases from poly(ADP-ribosyl)ation-induced inhibition. Hence, we purified and partially sequenced such a nuclease isolated from bovine seminal plasma and identified human, rat and mouse homologs of this enzyme. The extent of sequence homology among these nucleases indicates that these four proteins are orthologous members of the family of DNase I-related enzymes. We demonstrate that the activation of the human homolog previously specified as DNAS1L3 can induce Ca2+- and Mg2+-dependent DNA fragmentation in vitro and in vivo. RT-PCR analysis failed to detect DNAS1L3 mRNA in HeLa cells and nuclei isolated from these 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 fragmentation in the presence of both ions. The Ca2+ionophore ionomycin also induced internucleosomal DNA degradation in transfected but not in control HeLa cells. Transverse alternating field electrophoresis revealed that in nuclei from transfected HeLa cells, but not in those from control cells, DNA was 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 fragments into 50 kb and oligonucleosomal fragments. These results demonstrate that DNAS1L3 is necessary for Ca2+- and Mg2+-dependent cleavage of DNA into both oligonucleosomal and high molecular mass fragments in specific cell types.
钙和镁依赖性核酸内切酶与细胞凋亡过程中的DNA片段化有关。我们已经证明，这类特定的核酸酶会受到多聚（ADP-核糖基）化的抑制，并提出随后半胱天冬酶-3对PARP的切割可能会使这些核酸酶从多聚（ADP-核糖基）化诱导的抑制中释放出来。因此，我们纯化了从牛精浆中分离出的这种核酸酶并进行了部分测序，鉴定出了该酶的人、大鼠和小鼠同源物。这些核酸酶之间的序列同源程度表明，这四种蛋白质是DNA酶I相关酶家族的直系同源成员。我们证明，先前指定为DNAS1L3的人同源物的激活可在体外和体内诱导钙和镁依赖性的DNA片段化。RT-PCR分析未能在HeLa细胞中检测到DNAS1L3 mRNA，并且从这些细胞中分离出的细胞核在钙和镁存在下孵育时未表现出核小体间DNA片段化。然而，从稳定转染了DNAS1L3 cDNA的HeLa细胞中分离出的细胞核在两种离子存在下都发生了这种DNA片段化。钙离子载体离子霉素也在转染的HeLa细胞中诱导了核小体间DNA降解，但在对照HeLa细胞中未诱导。横向交变电场电泳显示，在转染的HeLa细胞的细胞核中，而不是对照细胞的细胞核中，DNA在镁存在下被切割成大于1000 kb的片段；在镁存在下添加钙导致大于1000 kb的片段加工成50 kb和寡核小体片段。这些结果表明，DNAS1L3对于在特定细胞类型中将DNA钙和镁依赖性切割成寡核小体和高分子量片段是必需的。
