Daley James M, Chiba Tamara, Xue Xiaoyu, Niu Hengyao, Sung Patrick
Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT 06520, USA.
Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT 06520, USA
Nucleic Acids Res. 2014;42(17):11083-91. doi: 10.1093/nar/gku803. Epub 2014 Sep 8.
BLM, a RecQ family DNA helicase mutated in Bloom's Syndrome, participates in homologous recombination at two stages: 5' DNA end resection and double Holliday junction dissolution. BLM exists in a complex with Topo IIIα, RMI1 and RMI2. Herein, we address the role of Topo IIIα and RMI1-RMI2 in resection using a reconstituted system with purified human proteins. We show that Topo IIIα stimulates DNA unwinding by BLM in a manner that is potentiated by RMI1-RMI2, and that the processivity of resection is reliant on the Topo IIIα-RMI1-RMI2 complex. Topo IIIα localizes to the ends of double-strand breaks, thus implicating it in the recruitment of resection factors. While the single-stranded DNA binding protein RPA plays a major role in imposing the 5' to 3' polarity of resection, Topo IIIα also makes a contribution in this regard. Moreover, we show that DNA2 stimulates the helicase activity of BLM. Our results thus uncover a multifaceted role of the Topo IIIα-RMI1-RMI2 ensemble and of DNA2 in the DNA resection reaction.
BLM是一种在布卢姆综合征中发生突变的RecQ家族DNA解旋酶，在同源重组的两个阶段发挥作用：5'端DNA切除和双Holliday连接解离。BLM与拓扑异构酶IIIα、RMI1和RMI2形成复合物存在。在此，我们使用纯化的人类蛋白重组系统来研究拓扑异构酶IIIα和RMI1-RMI2在切除过程中的作用。我们发现拓扑异构酶IIIα以一种被RMI1-RMI2增强的方式刺激BLM进行DNA解旋，并且切除的持续性依赖于拓扑异构酶IIIα-RMI1-RMI2复合物。拓扑异构酶IIIα定位于双链断裂的末端，因此表明它参与了切除因子的募集。虽然单链DNA结合蛋白RPA在赋予切除的5'到3'极性方面起主要作用，但拓扑异构酶IIIα在这方面也有贡献。此外，我们表明DNA2刺激BLM的解旋酶活性。因此，我们的结果揭示了拓扑异构酶IIIα-RMI1-RMI2组合体和DNA2在DNA切除反应中的多方面作用。
