Lévy Nicolas, Martz Adeline, Bresson Anne, Spenlehauer Catherine, de Murcia Gilbert, Ménissier-de Murcia Josiane
Département Intégrité du génome de l'UMR 7175, CNRS, Ecole Supérieure de Biotechnologie de Strasbourg, Boulevard S. Brant, BP 10413, F-67412 Illkirch Cedex, France.
Nucleic Acids Res. 2006 Jan 5;34(1):32-41. doi: 10.1093/nar/gkj409. Print 2006.
The two BRCT domains (BRCT1 and BRCT2) of XRCC1 mediate a network of protein-protein interactions with several key factors of the DNA single-strand breaks (SSBs) and base damage repair pathways. BRCT1 is required for the immediate poly(ADP-ribose)-dependent recruitment of XRCC1 to DNA breaks and is essential for survival after DNA damage. To better understand the biological role of XRCC1 in the processing of DNA ends, a search for the BRCT1 domain-associated proteins was performed by mass spectrometry of GST-BRCT1 pulled-down proteins from HeLa cell extracts. Here, we report that the double-strand break (DSB) repair heterotrimeric complex DNA-PK interacts with the BRCT1 domain of XRCC1 and phosphorylates this domain at serine 371 after ionizing irradiation. This caused XRCC1 dimer dissociation. The XRCC1 R399Q variant allele did not affect this phosphorylation. We also show that XRCC1 strongly stimulates the phosphorylation of p53-Ser15 by DNA-PK. The pseudo phosphorylated S371D mutant was a much weaker stimulator of DNA-PK activity whereas the non-phosphorylable mutant S371L endowed with a DNA-PK stimulating capacity failed to fully rescue the DSB repair defect of XRCC1-deficient EM9 rodent cells. The functional association between XRCC1 and DNA-PK in response to IR provides the first evidence for their involvement in a common DSB repair pathway.
XRCC1的两个BRCT结构域（BRCT1和BRCT2）介导了与DNA单链断裂（SSB）和碱基损伤修复途径的几个关键因子的蛋白质-蛋白质相互作用网络。BRCT1是XRCC1立即依赖多聚（ADP-核糖）招募到DNA断裂处所必需的，并且对于DNA损伤后的存活至关重要。为了更好地理解XRCC1在DNA末端加工中的生物学作用，通过对来自HeLa细胞提取物的GST-BRCT1下拉蛋白进行质谱分析，寻找与BRCT1结构域相关的蛋白质。在此，我们报告双链断裂（DSB）修复异源三聚体复合物DNA-PK与XRCC1的BRCT1结构域相互作用，并在电离辐射后使该结构域的丝氨酸371磷酸化。这导致XRCC1二聚体解离。XRCC1 R399Q变异等位基因不影响这种磷酸化。我们还表明，XRCC1强烈刺激DNA-PK对p53-Ser15的磷酸化。假磷酸化的S371D突变体对DNA-PK活性的刺激作用要弱得多，而具有DNA-PK刺激能力的不可磷酸化突变体S371L未能完全挽救XRCC1缺陷的EM9啮齿动物细胞的DSB修复缺陷。XRCC1和DNA-PK在响应IR时的功能关联为它们参与共同的DSB修复途径提供了首个证据。
