Kinetics of presynaptic filament assembly in the presence of single-stranded DNA binding protein and recombination mediator protein.
Kinetics of presynaptic filament assembly in the presence of single-stranded DNA binding protein and recombination mediator protein.
复制标题
单链 DNA 结合蛋白和重组介体蛋白存在下突触前丝组装的动力学。
DOI：
10.1021/bi401060p
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发表时间：
2013
期刊：
影响因子：
2.9
通讯作者：
Morrical,ScottW
中科院分区：
文献类型：
--
作者：
Liu,Jie;Berger,ChristopherL;Morrical,ScottW
RecA/RAD51家族的酶催化DNA链交换反应，这对于同源重组和DNA双链断裂的准确修复是重要的。RecA/RAD51重组酶通过组装成单链DNA(SsDNA)上的突触前细丝而被激活，这一过程受ssDNA结合蛋白(SSB)和中介蛋白的调节。中介蛋白通过加速进入的重组酶对SSB从单链DNA中限速置换来刺激链交换。在重组中，使用介体是一种高度保守的策略，但介体活性的确切机制尚不清楚。本研究利用成熟的T4噬菌体重组系统(UvsX重组酶、Gp32SSB和UvsY介体)，在体外研究了天然单链DNA上突触前微丝组装的动力学。结果表明，在没有介体的情况下，UvsX突触前丝在Gp32覆盖的单链DNA上依赖于ATP的组装受到盐敏感成核步骤的限制。丝状核化被选择性地增强，并由中介蛋白UvsY提供耐盐性，它似乎稳定了核化前的复合体。这一机制潜在地解释了UvsY如何在生理相关的离子强度和Gp32浓度下促进突触前细丝组装。其他数据表明，突触前微丝组装涉及多个成核事件，导致许多短小的UvsX-ssDNA微丝或簇，这可能是体内重组的相关形式。总之，这些发现提供了第一个详细的突触前细丝组装动力学模型，涉及到天然单链DNA上所有三个主要蛋白质组分(重组酶、介体和SSB)。
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 ssDNAin 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 recombinationin 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.