Yu Yaping, Wang Wei, Ding Qi, Ye Ruiqiong, Chen Dawn, Merkle Dennis, Schriemer David, Meek Katheryn, Lees-Miller Susan P
Department of Biochemistry & Molecular Biology, University of Calgary, 3330 Hospital Drive, NW, Calgary, Alta, Canada T2N 1N4.
DNA Repair (Amst). 2003 Nov 21;2(11):1239-52. doi: 10.1016/s1568-7864(03)00143-5.
Nonhomologous end joining (NHEJ) is a major pathway for the repair of DNA double-strand breaks (DSBs) in higher eukaryotes. Several proteins, including the DNA-dependent protein kinase (DNA-PK), XRCC4 and DNA ligase IV, are required for nonhomologous end joining both in vitro and in vivo. Since XRCC4 is recruited to the DNA double-strand break with DNA-PK, and because the protein kinase activity of DNA-PK is required for its in vivo function, we reasoned that XRCC4 could be a potential physiological substrate of DNA-PK. Here, we have used mass spectrometry to map the DNA-PK phosphorylation sites in XRCC4. Two major phosphorylation sites (serines 260 and 318), as well as several minor sites were identified. All of the identified sites lie within the carboxy-terminal 100 amino acids of XRCC4. Substitution of each of these sites to alanine (in combination) reduced the ability of DNA-PK to phosphorylate XRCC4 in vitro by at least two orders of magnitude. However, XRCC4-deficient cells that were complemented with XRCC4 lacking DNA-PK phosphorylation sites were analogous to wild type XRCC4 with respect to survival after ionizing radiation and ability to repair DSBs introduced during V(D)J recombination.
非同源末端连接（NHEJ）是高等真核生物中DNA双链断裂（DSB）修复的主要途径。包括DNA依赖性蛋白激酶（DNA-PK）、XRCC4和DNA连接酶IV在内的几种蛋白质在体外和体内的非同源末端连接中都是必需的。由于XRCC4与DNA-PK一起被招募到DNA双链断裂处，并且由于DNA-PK的蛋白激酶活性是其体内功能所必需的，我们推断XRCC4可能是DNA-PK的潜在生理底物。在这里，我们使用质谱法绘制了XRCC4中DNA-PK的磷酸化位点。确定了两个主要磷酸化位点（丝氨酸260和318）以及几个次要位点。所有已确定的位点都位于XRCC4的羧基末端100个氨基酸内。将这些位点中的每一个替换为丙氨酸（组合）可使DNA-PK在体外磷酸化XRCC4的能力降低至少两个数量级。然而，用缺乏DNA-PK磷酸化位点的XRCC4互补的XRCC4缺陷细胞在电离辐射后的存活率和修复V（D）J重组过程中引入的DSB的能力方面与野生型XRCC4相似。
