Perrault Ronel, Wang Huichen, Wang Minli, Rosidi Bustanur, Iliakis George
Department of Radiation Oncology, Division of Experimental Radiation Oncology, Kimmel Cancer Center, Jefferson Medical College, Philadelphia, Pennsylvania 19107, USA.
J Cell Biochem. 2004 Jul 1;92(4):781-94. doi: 10.1002/jcb.20104.
In cells of higher eukaryotes double strand breaks (DSBs) induced in the DNA after exposure to ionizing radiation (IR) are rapidly rejoined by a pathway of non-homologous end joining (NHEJ) that requires DNA dependent protein kinase (DNA-PK) and is therefore termed here D-NHEJ. When this pathway is chemically or genetically inactivated, cells still remove the majority of DSBs using an alternative, backup pathway operating independently of the RAD52 epistasis group of genes and with an order of magnitude slower kinetics (B-NHEJ). Here, we investigate the role of DNA-PK in the functional coordination of D-NHEJ and B-NHEJ using as a model end joining by cell extracts of restriction endonuclease linearized plasmid DNA. Although DNA end joining is inhibited by wortmannin, an inhibitor of DNA-PK, the degree of inhibition depends on the ratio between DNA ends and DNA-PK, suggesting that binding of inactive DNA-PK to DNA ends not only blocks processing by D-NHEJ, but also prevents the function of B-NHEJ. Residual end joining under conditions of incomplete inhibition, or in cells lacking DNA-PK, is attributed to the function of B-NHEJ operating on DNA ends free of DNA-PK. Thus, DNA-PK suppresses alternative pathways of end joining by efficiently binding DNA ends and shunting them to D-NHEJ.
在高等真核生物细胞中，暴露于电离辐射（IR）后DNA中诱导产生的双链断裂（DSB）通过非同源末端连接（NHEJ）途径迅速重新连接，该途径需要DNA依赖性蛋白激酶（DNA-PK），因此在这里被称为D-NHEJ。当该途径在化学上或基因上失活时，细胞仍然使用一种独立于RAD52上位性基因群运行且动力学速度慢一个数量级的替代备用途径（B-NHEJ）来去除大部分DSB。在这里，我们以限制性内切酶线性化质粒DNA的细胞提取物进行末端连接为模型，研究DNA-PK在D-NHEJ和B-NHEJ功能协调中的作用。尽管DNA末端连接受到DNA-PK抑制剂渥曼青霉素的抑制，但抑制程度取决于DNA末端与DNA-PK之间的比例，这表明无活性的DNA-PK与DNA末端的结合不仅会阻断D-NHEJ的加工过程，还会阻止B-NHEJ的功能。在不完全抑制条件下或在缺乏DNA-PK的细胞中的残余末端连接，归因于B-NHEJ对不含DNA-PK的DNA末端的作用。因此，DNA-PK通过有效结合DNA末端并将它们分流到D-NHEJ来抑制末端连接的替代途径。
