Leber R, Wise T W, Mizuta R, Meek K
Harold C. Simmons Arthritis Research Center, University of Texas Southwestern Medical Center, Dallas 75235, USA.
J Biol Chem. 1998 Jan 16;273(3):1794-801. doi: 10.1074/jbc.273.3.1794.
The gene product of XRCC4 has been implicated in both V(D)J recombination and the more general process of double strand break repair (DSBR). To date its role in these processes is unknown. Here, we describe biochemical characteristics of the murine XRCC4 protein. XRCC4 expressed in insect cells exists primarily as a disulfide-linked homodimer, although it can also form large multimers. Recombinant XRCC4 is phosphorylated during expression in insect cells. XRCC4 phosphorylation in Sf9 cells occurs on serine, threonine, and tyrosine residues. We also investigated whether XRCC4 interacts with the other factor known to be requisite for both V(D)J recombination and DSBR, the DNA-dependent protein kinase. We report that XRCC4 is an efficient in vitro substrate of DNA-PK and another unidentified serine/ threonine protein kinase(s). Both DNA-PK dependent and independent phosphorylation of XRCC4 in vitro occurs only on serine and threonine residues within the COOH-terminal 130 amino acids, a region of the molecule that is not absolutely required for XRCC4's DSBR function. Finally, recombinant XRCC4 facilitates Ku binding to DNA, promoting assembly of DNA-PK and complexing with DNA-PK bound to DNA. These data are consistent with the hypothesis that XRCC4 functions as an alignment factor in the DNA-PK complex.
XRCC4的基因产物与V(D)J重组以及更普遍的双链断裂修复(DSBR)过程都有关联。迄今为止，其在这些过程中的作用尚不清楚。在此，我们描述了小鼠XRCC4蛋白的生化特性。在昆虫细胞中表达的XRCC4主要以二硫键连接的同二聚体形式存在，尽管它也能形成大型多聚体。重组XRCC4在昆虫细胞表达过程中会发生磷酸化。Sf9细胞中的XRCC4磷酸化发生在丝氨酸、苏氨酸和酪氨酸残基上。我们还研究了XRCC4是否与另一种已知对V(D)J重组和DSBR都必需的因子——DNA依赖性蛋白激酶相互作用。我们报告称，XRCC4是DNA-PK和另一种未鉴定的丝氨酸/苏氨酸蛋白激酶在体外的有效底物。XRCC4在体外的DNA-PK依赖性和非依赖性磷酸化仅发生在COOH末端130个氨基酸内的丝氨酸和苏氨酸残基上，该分子区域对于XRCC4的DSBR功能并非绝对必需。最后，重组XRCC4促进Ku与DNA结合，促进DNA-PK的组装并与结合在DNA上的DNA-PK形成复合物。这些数据与XRCC4在DNA-PK复合物中作为排列因子发挥作用的假设一致。
