UMR5240, Microbiologie, Adaptation et Pathogénie, University of Lyon, Université Claude Bernard Lyon 1, INSA-Lyon, CNRS, 11 Avenue Jean Capelle, 69621, Villeurbanne, France.
Molecular Innovation Unit, Centre Christophe Mérieux, bioMérieux, 5 Rue des Berges, 38024, Grenoble Cedex 01, France.
Sci Rep. 2021 Oct 26;11(1):21106. doi: 10.1038/s41598-021-00589-9.
RecA plays a central role in DNA repair and is a main actor involved in recombination and activation of the SOS response. It is also used in the context of biotechnological applications in recombinase polymerase isothermal amplification (RPA). In this work, we studied the biological properties of seven RecA variants, in particular their recombinogenic activity and their ability to induce the SOS response, to better understand the structure-function relationship of RecA and the effect of combined mutations. We also investigated the biochemical properties of RecA variants that may be useful for the development of biotechnological applications. We showed that Dickeya dadantii RecA (DdRecA) had an optimum strand exchange activity at 30 °C and in the presence of a dNTP mixture that inhibited Escherichia coli RecA (EcRecA). The differences between the CTD and C-tail of the EcRecA and DdRecA domains could explain the altered behaviour of DdRecA. D. radiodurans RecA (DrRecA) was unable to perform recombination and activation of the SOS response in an E. coli context, probably due to its inability to interact with E. coli recombination accessory proteins and SOS LexA repressor. DrRecA strand exchange activity was totally inhibited in the presence of chloride ions but worked well in acetate buffer. The overproduction of Pseudomonas aeruginosa RecA (PaRecA) in an E. coli context was responsible for a higher SOS response and defects in cellular growth. PaRecA was less inhibited by the dNTP mixture than EcRecA. Finally, the study of three variants, namely, EcPa, EcRecAV1 and EcRecAV2, that contained a combination of mutations that, taken independently, are described as improving recombination, led us to raise new hypotheses on the structure-function relationship and on the monomer-monomer interactions that perturb the activity of the protein as a whole.
RecA 在 DNA 修复中起着核心作用，是涉及重组和激活 SOS 反应的主要参与者。它也用于生物技术应用中的重组酶聚合等温扩增（RPA）。在这项工作中，我们研究了七种 RecA 变体的生物学特性，特别是它们的重组活性和诱导 SOS 反应的能力，以更好地理解 RecA 的结构-功能关系和组合突变的影响。我们还研究了可能对生物技术应用的发展有用的 RecA 变体的生化特性。我们表明，Dickeya dadantii RecA（DdRecA）在 30°C 和含有抑制大肠杆菌 RecA（EcRecA）的 dNTP 混合物的条件下具有最佳的链交换活性。EcRecA 和 DdRecA 结构域的 CTD 和 C-尾之间的差异可以解释 DdRecA 的改变行为。D. radiodurans RecA（DrRecA）在大肠杆菌背景下无法进行重组和激活 SOS 反应，可能是由于其无法与大肠杆菌重组辅助蛋白和 SOS LexA 阻遏物相互作用。氯离子的存在完全抑制了 DrRecA 的链交换活性，但在乙酸盐缓冲液中效果良好。大肠杆菌背景下过量产生的铜绿假单胞菌 RecA（PaRecA）导致 SOS 反应更高和细胞生长缺陷。与 EcRecA 相比，PaRecA 受 dNTP 混合物的抑制作用较小。最后，对三种变体，即 EcPa、EcRecAV1 和 EcRecAV2 的研究表明，它们包含的突变组合，单独来看，可提高重组效率，这使我们对结构-功能关系以及单体-单体相互作用提出了新的假设，这些相互作用会干扰蛋白质的整体活性。
