Comprehensive analysis of recombination auxiliary factors

DNA双链断裂(DSB)是DNA损伤最严重的形式。同源重组(HR)是一种利用位于基因组其他地方的相同遗传信息作为模板进行准确DSB修复的机制。RAD51是HR的核心蛋白，但参与RAD51调控的辅助因子有RAD52、RAD54、RAD55-RAD57和Swi5-Sfr1。这些辅助因子都与RAD51结合，这种结合被认为是它们在RAD51增强中的关键作用。尽管如此，人们对这些相互作用的性质知之甚少，特别是关于Rad55-Rad57。我们最初试图对Rad55-Rad57进行生化表征。虽然我们成功地进行了纯化，但由于Rad55-Rad57的生物化学难解性，我们无法深入表征相互作用。因此，我们采用了一种不同的方法。RAD51的结构分析突出了一个暴露在溶剂中的突出酸性斑块(PAP)，由三个残基组成：E205，E206，D209。我们的基因分析表明，E206A突变特异性地损害了与Rad55-Rad57的相互作用。此外，三个残基均突变为Ala(RAD51-EED)，阻断了与Rad52的相互作用，进一步削弱了与Rad55-Rad57的相互作用，甚至削弱了与Rad54的相互作用。这些结果表明，PAP是一个新发现的RAD51基序，它是多种辅助因子相互作用的中心，促进了RAD51的增强。这些结果为辅助因素对HR的调节提供了重要的新见解。

DNA double-strand breaks (DSBs) are the severest form of DNA damage. Homologous recombination (HR) is a mechanism that utilizes identical genetic information located elsewhere in the genome as a template for accurate DSB repair. Rad51 is the central protein in HR, but multiple auxiliary factors are involved in modulating Rad51, including Rad52, Rad54, Rad55-Rad57, and Swi5-Sfr1. These auxiliary factors all bind to Rad51, and this binding is thought to be critical for their role in Rad51 potentiation. Despite this, little is known about the nature of these interactions, especially with regards to Rad55-Rad57.We originally tried to biochemically characterize Rad55-Rad57. Although we succeeded in purification, we were unable to characterize the interaction in-depth due to the biochemical intractability of Rad55-Rad57. We therefore employed a different approach. Structural analysis of Rad51 highlighted a solvent-exposed protruding acidic patch (PAP) comprised of three residues: E205, E206, D209. Our genetic analysis demonstrated that the E206A mutation specifically impairs the interaction with Rad55-Rad57. Moreover, mutation of all three residues to Ala (Rad51-EED) abrogated the interaction with Rad52, further impaired the interaction with Rad55-Rad57, and even impaired the interaction with Rad54. These results demonstrate that the PAP, a newly identified motif of Rad51, functions as an interaction hub for multiple auxiliary factors to facilitate the potentiation of Rad51. These results provided critical new insights into the regulation of HR by auxiliary factors.