Kar Ananya, Kaur Manpreet, Ghosh Tanushree, Khan Md Muntaz, Sharma Aparna, Shekhar Ritu, Varshney Akhil, Saxena Sandeep
National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi-110067, India.
National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi-110067, India
Nucleic Acids Res. 2015 May 26;43(10):4962-74. doi: 10.1093/nar/gkv369. Epub 2015 Apr 27.
The primary eukaryotic single-stranded DNA-binding protein, Replication protein A (RPA), binds to single-stranded DNA at the sites of DNA damage and recruits the apical checkpoint kinase, ATR via its partner protein, ATRIP. It has been demonstrated that absence of RPA incapacitates the ATR-mediated checkpoint response. We report that in the absence of RPA, human single-stranded DNA-binding protein 1 (hSSB1) and its partner protein INTS3 form sub-nuclear foci, associate with the ATR-ATRIP complex and recruit it to the sites of genomic stress. The ATRIP foci formed after RPA depletion are abrogated in the absence of INTS3, establishing that hSSB-INTS3 complex recruits the ATR-ATRIP checkpoint complex to the sites of genomic stress. Depletion of homologs hSSB1/2 and INTS3 in RPA-deficient cells attenuates Chk1 phosphorylation, indicating that the cells are debilitated in responding to stress. We have identified that TopBP1 and the Rad9-Rad1-Hus1 complex are essential for the alternate mode of ATR activation. In summation, we report that the single-stranded DNA-binding protein complex, hSSB1/2-INTS3 can recruit the checkpoint complex to initiate ATR signaling.
主要的真核生物单链DNA结合蛋白，即复制蛋白A（RPA），在DNA损伤位点与单链DNA结合，并通过其伴侣蛋白ATR相互作用蛋白（ATRIP）招募顶端检查点激酶ATR。已有研究表明，缺乏RPA会使ATR介导的检查点反应丧失功能。我们报告称，在缺乏RPA的情况下，人类单链DNA结合蛋白1（hSSB1）及其伴侣蛋白INTS3会形成亚核灶，与ATR-ATRIP复合物结合，并将其招募到基因组应激位点。在缺乏INTS3的情况下，RPA耗竭后形成的ATRIP灶消失，这表明hSSB-INTS3复合物将ATR-ATRIP检查点复合物招募到基因组应激位点。在RPA缺陷细胞中，同源物hSSB1/2和INTS3的耗竭会减弱Chk1磷酸化，表明细胞在应对应激时功能受损。我们已经确定，TopBP1和Rad9-Rad1-Hus1复合物对于ATR激活的替代模式至关重要。总之，我们报告称，单链DNA结合蛋白复合物hSSB1/2-INTS3可以招募检查点复合物以启动ATR信号传导。
