Kawane Kohki, Nagata Shigekazu
Department of Medical Chemistry, Graduate School of Medicine, Kyoto University, Yoshida, Sakyo-ku, Kyoto, Japan.
Methods Enzymol. 2008;442:271-87. doi: 10.1016/S0076-6879(08)01414-6.
DNA degradation is one of the hallmarks of programmed cell death, or apoptosis. Recent analyses of this process revealed that apoptotic DNA degradation is mediated by two independent mechanisms. First, the caspase-activated DNase (CAD) cell autonomously cleaves DNA into nucleosomal units in dying cells. Then, after the apoptotic cells are engulfed by macrophages, the fragmented DNA is further degraded by DNase II in the lysosomes of the macrophages. This chapter describes assay procedures for CAD and DNase II. It includes biochemical methods for quantifying DNase activity and cell culture systems to follow cell-autonomous and noncell-autonomous DNA degradation. These techniques are useful for studying DNases that are involved in programmed cell death and for following the engulfment of apoptotic cells by phagocytes.
DNA降解是程序性细胞死亡（即凋亡）的标志之一。最近对这一过程的分析表明，凋亡性DNA降解由两种独立机制介导。首先，半胱天冬酶激活的脱氧核糖核酸酶（CAD）在垂死细胞中自主地将DNA切割成核小体单位。然后，凋亡细胞被巨噬细胞吞噬后，破碎的DNA在巨噬细胞的溶酶体中被脱氧核糖核酸酶II进一步降解。本章描述了CAD和脱氧核糖核酸酶II的检测程序。它包括用于定量脱氧核糖核酸酶活性的生化方法以及用于追踪细胞自主和非细胞自主DNA降解的细胞培养系统。这些技术对于研究参与程序性细胞死亡的脱氧核糖核酸酶以及追踪吞噬细胞对凋亡细胞的吞噬作用很有用。
