Role of DNAS1L3 in Ca2+- and Mg2+-dependent cleavage of DNA into oligonucleosomal and high molecular mass fragments.
Role of DNAS1L3 in Ca2+- and Mg2+-dependent cleavage of DNA into oligonucleosomal and high molecular mass fragments.
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DOI：
10.1093/nar/27.9.1999
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发表时间：
1999-05
影响因子：
14.9
通讯作者：
Alexander G. Yakovlev;Geping Wang;Bogdan A. Stoica;C. Simbulan-Rosenthal;K. Yoshihara;M. Smulson
中科院分区：
文献类型：
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作者：
Alexander G. Yakovlev;Geping Wang;Bogdan A. Stoica;C. Simbulan-Rosenthal;K. Yoshihara;M. Smulson
Ca 2+和Mg 2+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，这种类型的特定核酸酶被聚（ADP-核糖基）化抑制，并建议随后由半胱天冬酶-3切割PARP可能释放这些核酸酶从聚（ADP-核糖基）化诱导的抑制。因此，我们纯化和部分测序这样的核酸酶从牛精浆中分离，并确定人类，大鼠和小鼠的同源酶。这些核酸酶之间的序列同源性的程度表明，这四种蛋白质是DNA酶I相关酶家族的直系成员。我们证明，激活的人同系物以前指定为DNAS 1 L3可以诱导钙和镁离子依赖性DNA片段在体外和体内。RT-PCR分析未能检测到HeLa细胞中的DNAS 1 L3 mRNA，并且当在Ca 2+和Mg 2+存在下孵育时，从这些细胞中分离的细胞核没有表现出核小体间DNA片段化。然而，从已经用DNAS 1 L3 cDNA稳定转染的HeLa细胞中分离的细胞核在两种离子存在下经历这样的DNA片段化。Ca 2+离子载体离子霉素也诱导核小体间DNA降解转染，但不是在控制HeLa细胞。横向交变场电泳显示，在转染的HeLa细胞的细胞核中，而不是在对照细胞的细胞核中，DNA在Mg 2+存在下被切割成>1000 kb的片段;在Mg 2+存在下加入Ca 2+导致>1000 kb的片段被加工成50 kb和寡核小体片段。这些结果表明，DNAS 1 L3是必要的钙和镁2+依赖性的DNA切割成寡核小体和高分子量片段在特定的细胞类型。
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.