Department of Pharmacology & Toxicology, University of Arkansas for Medical Sciences, 4301 West Markham Street, #638, Little Rock, AR 72205, USA.
Central Arkansas Veterans Healthcare System, 4300 West 7th Street, Little Rock, AR 72205, USA.
Int J Mol Sci. 2020 Nov 17;21(22):8665. doi: 10.3390/ijms21228665.
Endonuclease-mediated DNA fragmentation is both an immediate cause and a result of apoptosis and of all other types of irreversible cell death after injury. It is produced by nine enzymes including DNase I, DNase 2, their homologs, caspase-activated DNase (CAD) and endonuclease G (EndoG). The endonucleases act simultaneously during cell death; however, regulatory links between these enzymes have not been established. We hypothesized that DNase I, the most abundant of endonucleases, may regulate other endonucleases. To test this hypothesis, rat kidney tubular epithelial NRK-52E cells were transfected with the DNase I gene or its inactive mutant in a pECFP expression vector, while control cells were transfected with the empty vector. mRNA expression of all nine endonucleases was studied using real-time RT-PCR; DNA strand breaks in endonuclease genes were determined by PCR and protein expression of the enzymes was measured by Western blotting and quantitative immunocytochemistry. Our data showed that DNase I, but not its inactive mutant, induces all other endonucleases at varying time periods after transfection, causes DNA breaks in endonuclease genes, and elevates protein expression of several endonucleases. This is the first evidence that endonucleases seem to be induced by the DNA-degrading activity of DNase I.
核酸内切酶介导的 DNA 片段化既是细胞凋亡和损伤后所有其他类型不可逆细胞死亡的直接原因，也是其结果。它由包括 DNase I、DNase 2、它们的同源物、半胱天冬酶激活的核酸内切酶 (CAD) 和核酸内切酶 G (EndoG) 在内的 9 种酶产生。这些核酸内切酶在细胞死亡过程中同时发挥作用；然而，这些酶之间的调节联系尚未建立。我们假设，DNase I 作为最丰富的核酸内切酶之一，可能调节其他核酸内切酶。为了验证这一假设，我们将 DNase I 基因或其无活性突变体转染到 pECFP 表达载体中，而对照细胞则转染空载体，转染到大鼠肾管状上皮 NRK-52E 细胞中。使用实时 RT-PCR 研究了所有 9 种核酸内切酶的 mRNA 表达；通过 PCR 测定核酸内切酶基因中的 DNA 链断裂，通过 Western blot 和定量免疫细胞化学测定酶的蛋白表达。我们的数据表明，DNase I（而非其无活性突变体）在转染后不同时间诱导所有其他核酸内切酶，导致核酸内切酶基因中的 DNA 断裂，并提高几种核酸内切酶的蛋白表达。这是第一个证据表明核酸内切酶似乎是由 DNA 降解酶活性的 DNase I 诱导的。
