Shell Steven M, Li Zhengke, Shkriabai Nikolozi, Kvaratskhelia Mamuka, Brosey Chris, Serrano Moises A, Chazin Walter J, Musich Phillip R, Zou Yue
Department of Biochemistry and Molecular Biology, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, Tennessee 37614, USA.
J Biol Chem. 2009 Sep 4;284(36):24213-22. doi: 10.1074/jbc.M109.000745. Epub 2009 Jul 8.
In response to DNA damage, eukaryotic cells activate a series of DNA damage-dependent pathways that serve to arrest cell cycle progression and remove DNA damage. Coordination of cell cycle arrest and damage repair is critical for maintenance of genomic stability. However, this process is still poorly understood. Nucleotide excision repair (NER) and the ATR-dependent cell cycle checkpoint are the major pathways responsible for repair of UV-induced DNA damage. Here we show that ATR physically interacts with the NER factor Xeroderma pigmentosum group A (XPA). Using a mass spectrometry-based protein footprinting method, we found that ATR interacts with a helix-turn-helix motif in the minimal DNA-binding domain of XPA where an ATR phosphorylation site (serine 196) is located. XPA-deficient cells complemented with XPA containing a point mutation of S196A displayed a reduced repair efficiency of cyclobutane pyrimidine dimers as compared with cells complemented with wild-type XPA, although no effect was observed for repair of (6-4) photoproducts. This suggests that the ATR-dependent phosphorylation of XPA may promote NER repair of persistent DNA damage. In addition, a K188A point mutation of XPA that disrupts the ATR-XPA interaction inhibits the nuclear import of XPA after UV irradiation and, thus, significantly reduced DNA repair efficiency. By contrast, the S196A mutation has no effect on XPA nuclear translocation. Taken together, our results suggest that the ATR-XPA interaction mediated by the helix-turn-helix motif of XPA plays an important role in DNA-damage responses to promote cell survival and genomic stability after UV irradiation.
为应对DNA损伤，真核细胞会激活一系列依赖于DNA损伤的通路，这些通路可使细胞周期进程停滞并去除DNA损伤。细胞周期停滞与损伤修复的协调对于维持基因组稳定性至关重要。然而，这一过程仍未被充分理解。核苷酸切除修复（NER）和ATR依赖的细胞周期检查点是负责修复紫外线诱导的DNA损伤的主要通路。在此，我们发现ATR与NER因子A型着色性干皮病蛋白（XPA）存在物理相互作用。使用基于质谱的蛋白质足迹法，我们发现ATR与XPA最小DNA结合结构域中的一个螺旋-转角-螺旋基序相互作用，ATR磷酸化位点（丝氨酸196）就位于该基序处。与用野生型XPA互补的细胞相比，用含有S196A点突变的XPA互补的XPA缺陷细胞对环丁烷嘧啶二聚体的修复效率降低，尽管对（6-4）光产物的修复未观察到影响。这表明ATR依赖的XPA磷酸化可能促进对持续性DNA损伤的NER修复。此外，破坏ATR-XPA相互作用的XPA的K188A点突变会抑制紫外线照射后XPA的核输入，从而显著降低DNA修复效率。相比之下，S196A突变对XPA核转运没有影响。综上所述，我们的结果表明，由XPA的螺旋-转角-螺旋基序介导的ATR-XPA相互作用在紫外线照射后的DNA损伤反应中发挥重要作用，以促进细胞存活和基因组稳定性。
