Department of Biochemistry and ‡Department of Molecular Physiology and Biophysics, University of Vermont College of Medicine , Burlington, Vermont 05405, United States.
Biochemistry. 2013 Nov 12;52(45):7878-89. doi: 10.1021/bi401060p. Epub 2013 Oct 30.
Enzymes of the RecA/Rad51 family catalyze DNA strand exchange reactions that are important for homologous recombination and for the accurate repair of DNA double-strand breaks. RecA/Rad51 recombinases are activated by their assembly into presynaptic filaments on single-stranded DNA (ssDNA), a process that is regulated by ssDNA binding protein (SSB) and mediator proteins. Mediator proteins stimulate strand exchange by accelerating the rate-limiting displacement of SSB from ssDNA by the incoming recombinase. The use of mediators is a highly conserved strategy in recombination, but the precise mechanism of mediator activity is unknown. In this study, the well-defined bacteriophage T4 recombination system (UvsX recombinase, Gp32 SSB, and UvsY mediator) is used to examine the kinetics of presynaptic filament assembly on native ssDNA in vitro. Results indicate that the ATP-dependent assembly of UvsX presynaptic filaments on Gp32-covered ssDNA is limited by a salt-sensitive nucleation step in the absence of mediator. Filament nucleation is selectively enhanced and rendered salt-resistant by mediator protein UvsY, which appears to stabilize a prenucleation complex. This mechanism potentially explains how UvsY promotes presynaptic filament assembly at physiologically relevant ionic strengths and Gp32 concentrations. Other data suggest that presynaptic filament assembly involves multiple nucleation events, resulting in many short UvsX-ssDNA filaments or clusters, which may be the relevant form for recombination in vivo. Together, these findings provide the first detailed kinetic model for presynaptic filament assembly involving all three major protein components (recombinase, mediator, and SSB) on native ssDNA.
RecA/Rad51 家族的酶催化 DNA 链交换反应，该反应对于同源重组和双链 DNA 断裂的准确修复很重要。RecA/Rad51 重组酶通过其在单链 DNA（ssDNA）上组装成预链交换体而被激活，该过程受 ssDNA 结合蛋白（SSB）和介质蛋白的调节。介质蛋白通过加速进入的重组酶将 SSB 从 ssDNA 上的限速置换来刺激链交换。在重组中使用介质蛋白是一种高度保守的策略，但介质蛋白活性的确切机制尚不清楚。在这项研究中，使用定义明确的噬菌体 T4 重组系统（UvsX 重组酶、Gp32 SSB 和 UvsY 介质）来体外检查天然 ssDNA 上预链交换体组装的动力学。结果表明，在没有介质的情况下，UvsX 预链交换体在 Gp32 覆盖的 ssDNA 上的 ATP 依赖性组装受到盐敏感性成核步骤的限制。介质蛋白 UvsY 选择性地增强并使成核过程具有抗盐性，这似乎稳定了预成核复合物。这种机制可能解释了 UvsY 如何在生理相关的离子强度和 Gp32 浓度下促进预链交换体的组装。其他数据表明，预链交换体的组装涉及多个成核事件，导致许多短的 UvsX-ssDNA 纤维或簇，这可能是体内重组的相关形式。这些发现共同提供了第一个涉及天然 ssDNA 上所有三个主要蛋白质成分（重组酶、介质和 SSB）的预链交换体组装的详细动力学模型。
