Burnham Daniel R, Nijholt Bas, De Vlaminck Iwijn, Quan Jinhua, Yusufzai Timur, Dekker Cees
Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, 2629 HZ, The Netherlands.
Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02215, USA.
Nucleic Acids Res. 2017 May 5;45(8):4687-4695. doi: 10.1093/nar/gkx147.
We investigate the mechanistic nature of the Snf2 family protein HARP, mutations of which are responsible for Schimke immuno-osseous dysplasia. Using a single-molecule magnetic tweezers assay, we construct RPA-stabilized DNA bubbles within torsionally constrained DNA to investigate the annealing action of HARP on a physiologically relevant substrate. We find that HARP closes RPA-stabilized bubbles in a slow reaction, taking on the order of tens of minutes for ∼600 bp of DNA to be re-annealed. The data indicate that DNA re-anneals through the removal of RPA, which is observed as clear steps in the bubble-closing traces. The dependence of the closing rate on both ionic strength and HARP concentration indicates that removal of RPA occurs via an association-dissociation mechanism where HARP does not remain associated with the DNA. The enzyme exhibits classical Michaelis-Menten kinetics and acts cooperatively with a Hill coefficient of 3 ± 1. Our work also allows the determination of some important features of RPA-bubble structures at low supercoiling, including the existence of multiple bubbles and that RPA molecules are mis-registered on the two strands.
我们研究了Snf2家族蛋白HARP的作用机制，该蛋白的突变会导致施姆克免疫性骨发育异常。我们使用单分子磁镊检测法，在受扭转约束的DNA中构建RPA稳定的DNA气泡，以研究HARP在生理相关底物上的退火作用。我们发现，HARP以缓慢的反应闭合RPA稳定的气泡，约600 bp的DNA重新退火需要几十分钟。数据表明，DNA通过去除RPA进行重新退火，这在气泡闭合轨迹中表现为明显的步骤。闭合速率对离子强度和HARP浓度的依赖性表明，RPA的去除是通过缔合-解离机制发生的，其中HARP不会与DNA保持结合。该酶表现出典型的米氏动力学，并以3±1的希尔系数协同作用。我们的工作还能够确定低超螺旋状态下RPA-气泡结构的一些重要特征，包括多个气泡的存在以及RPA分子在两条链上的错配。
