RPA resolves conflicting activities of accessory proteins during reconstitution of Dmc1-mediated meiotic recombination
RPA resolves conflicting activities of accessory proteins during reconstitution of Dmc1-mediated meiotic recombination
复制标题
DOI：
10.1093/nar/gky1160
复制
发表时间：
2019-01-25
影响因子：
14.9
通讯作者：
Bishop, Douglas K.
中科院分区：
文献类型：
--
作者：
Chan, Yuen-Ling;Zhang, Annie;Bishop, Douglas K.
Dmc1在出芽酵母和包括人类在内的许多其他生物的减数分裂重组过程中催化同源性搜索和链交换。本研究利用从出芽酵母中纯化的6种蛋白，包括Dmc1及其附属蛋白RPA、Rad51、Rdh54/Tid1、mei5 - sae3和Hop2-Mnd1，在体外重建Dmc1重组，以促进ssDNA和dsDNA底物之间d环的形成。每种辅助蛋白都对Dmc1的活性有贡献，所有六种蛋白的组合产生最佳活性。ssDNA结合蛋白RPA在刺激Dmc1活性方面发挥多种作用，包括克服ssDNA二级结构对d环反应的抑制作用，以及延长d环。此外，我们证明RPA限制了Hop2-Mnd1和Rdh54/Tid1的抑制相互作用，否则在Dmc1-ssDNA核蛋白丝的组装过程中会发生这种相互作用。最后，我们报道了生化重构中所用蛋白质之间的相互作用，包括Rad51和Dmc1之间的直接相互作用，该相互作用由Mei5-Sae3增强。
Dmc1 catalyzes homology search and strand exchange during meiotic recombination in budding yeast and many other organisms including humans. Here we reconstitute Dmc1 recombination in vitro using six purified proteins from budding yeast including Dmc1 and its accessory proteins RPA, Rad51, Rdh54/Tid1, Mei5-Sae3and Hop2-Mnd1 to promote D-loop formation between ssDNA and dsDNA substrates. Each accessory protein contributed to Dmc1's activity, with the combination of all six proteins yielding optimal activity. The ssDNA binding protein RPA plays multiple roles in stimulating Dmc1's activity including by overcoming inhibitory effects of ssDNA secondary structure on D-loop reactions, and by elongating D-loops. In addition, we demonstrate that RPA limits inhibitory interactions of Hop2-Mnd1 and Rdh54/Tid1 that otherwise occur during assembly of Dmc1-ssDNA nucleoprotein filaments. Finally, we report interactions between the proteins employed in the biochemical reconstitution including a direct interaction between Rad51 and Dmc1 that is enhanced by Mei5-Sae3.