DNA ligase III as a candidate component of backup pathways of nonhomologous end joining
DNA ligase III as a candidate component of backup pathways of nonhomologous end joining
复制标题
DOI：
10.1158/0008-5472.can-04-3055
复制
发表时间：
2005-05-15
期刊：
影响因子：
11.2
通讯作者：
Iliakis, G
中科院分区：
文献类型：
--
作者：
Wang, HC;Rosidi, B;Iliakis, G
生物化学和遗传学研究支持这样的观点，即电离辐射诱导的高等真核生物基因组中的大多数DNA双链断裂通过两种称为D-NHEJ和B-NHEJ的非同源末端连接（NHEJ）途径去除。然而D-NHEJ依赖于DNA依赖性蛋白激酶和DNA连接酶IV/XRCC 4的活性，B-NHEJ的组分尚未被鉴定。使用提取物分馏，我们表明，大多数的DNA末端连接活性的HeLa细胞提取物来自DNA连接酶III。DNA连接酶III通过两个柱分级分离，具有最大的DNA末端连接活性，并且其从提取物中的消耗导致活性损失，该活性损失可以通过添加纯化的酶来恢复。相同的分级分离方案提供了一个额外的因素的证据，强烈增强DNA末端连接和移动的产品谱从圆形多聚体。体内质粒测定显示，DNA连接酶IV缺陷型小鼠胚胎成纤维细胞保留显著的DNA末端连接活性，其可通过使用RNA干扰敲低DNA连接酶III而降低高达80%。这些体内和体外观察结果将DNA连接酶III鉴定为B-NHEJ的候选组分，并指出有助于NHEJ效率的其他因素。
Biochemical and genetic studies support the view that the majority of DNA double-strand breaks induced in the genome of higher eukaryotes by ionizing radiation are removed by two pathways of nonhomologous end joining (NHEJ) termed D-NHEJ and B-NHEJ. Whereas D-NHEJ depends on the activities of the DNA-dependent protein kinase and DNA ligase IV/XRCC4, components of B-NHEJ have not been identified. Using extract fractionation, we show that the majority of DNA end joining activity in extracts of HeLa cells derives from DNA ligase III. DNA ligase III fractionates through two columns with the maximum in DNA end joining activity and its depletion from the extract causes loss of activity that can be recovered by the addition of purified enzyme. The same fractionation protocols provide evidence for an additional factor strongly enhancing DNA end joining and shifting the product spectrum from circles to multimers. An in vivo plasmid assay shows that DNA ligase IV-deficient mouse embryo fibroblasts retain significant DNA end joining activity that can be reduced by up to 80% by knocking down DNA ligase III using RNA interference. These in vivo and in vitro observations identify DNA ligase III as a candidate component for B-NHEJ and point to additional factors contributing to NHEJ efficiency.