Grawunder U, Zimmer D, Kulesza P, Lieber M R
University of Southern California School of Medicine, Norris Comprehensive Cancer Center, Department of Pathology, Los Angeles, California 90033, USA.
J Biol Chem. 1998 Sep 18;273(38):24708-14. doi: 10.1074/jbc.273.38.24708.
The XRCC4 gene is required for the repair of DNA double-strand breaks in mammalian cells. Without XRCC4, cells are hypersensitive to ionizing radiation and deficient for V(D)J recombination. It has been demonstrated that XRCC4 binds and stimulates DNA ligase IV, which has led to the hypothesis that DNA ligase IV is essential for both of these processes. In this study deletion mutants of XRCC4 were tested for their ability to associate with DNA ligase IV in vitro and for their ability to reconstitute XRCC4-deficient cells in vivo. We find that a central region of XRCC4 from amino acids 100-250 is necessary for DNA ligase IV binding and that deletions within this region functionally inactivates XRCC4. Deletions within the C-terminal 84 amino acids neither affect DNA ligase IV binding nor the in vivo function of XRCC4. The correlation between the ability or inability of XRCC4 to bind DNA ligase IV and its ability or failure to reconstitute wild-type DNA repair in vivo, respectively, demonstrates for the first time that the physical interaction with DNA ligase IV is crucial for the in vivo function of XRCC4. Deletions within the N-terminal 100 amino acids inactivate XRCC4 in vivo but leave DNA ligase IV binding unaffected. This indicates further DNA ligase IV-independent functions of XRCC4.
XRCC4基因对于哺乳动物细胞中DNA双链断裂的修复是必需的。没有XRCC4，细胞对电离辐射高度敏感，并且V(D)J重组存在缺陷。已经证明XRCC4能结合并刺激DNA连接酶IV，这导致了DNA连接酶IV对于这两个过程都必不可少的假说。在本研究中，测试了XRCC4缺失突变体在体外与DNA连接酶IV结合的能力以及在体内重建XRCC4缺陷细胞的能力。我们发现，XRCC4中100-250位氨基酸的中央区域对于DNA连接酶IV的结合是必需的，并且该区域内的缺失会在功能上使XRCC4失活。C末端84个氨基酸内的缺失既不影响DNA连接酶IV的结合，也不影响XRCC4的体内功能。XRCC4与DNA连接酶IV结合与否的能力分别与其在体内重建野生型DNA修复的能力或失败之间的相关性，首次证明了与DNA连接酶IV的物理相互作用对于XRCC4的体内功能至关重要。N末端100个氨基酸内的缺失在体内使XRCC4失活，但不影响DNA连接酶IV的结合。这进一步表明了XRCC4具有不依赖DNA连接酶IV的功能。
