Graduate Group in Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania19104, United States.
Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania19104, United States.
Biochemistry. 2022 Dec 20;61(24):2884-2896. doi: 10.1021/acs.biochem.2c00505. Epub 2022 Dec 6.
The SOS response is a bacterial DNA damage response pathway that has been heavily implicated in bacteria's ability to evolve resistance to antibiotics. Activation of the SOS response is dependent on the interaction between two bacterial proteins, RecA and LexA. RecA acts as a DNA damage sensor by forming lengthy oligomeric filaments (RecA*) along single-stranded DNA (ssDNA) in an ATP-dependent manner. RecA* can then bind to LexA, the repressor of SOS response genes, triggering LexA degradation and leading to induction of the SOS response. Formation of the RecA*-LexA complex therefore serves as the key "SOS activation signal." Given the challenges associated with studying a complex involving multiple macromolecular interactions, the essential constituents of RecA* that allow LexA cleavage are not well defined. Here, we leverage head-to-tail linked and end-capped RecA constructs as tools to define the minimal RecA* filament that can engage LexA. In contrast to previously postulated models, we found that as few as three linked RecA units are capable of ssDNA binding, LexA binding, and LexA cleavage. We further demonstrate that RecA oligomerization alone is insufficient for LexA cleavage, with an obligate requirement for ATP and ssDNA binding to form a competent SOS activation signal with the linked constructs. Our minimal system for RecA* highlights the limitations of prior models for the SOS activation signal and offers a novel tool that can inform efforts to slow acquired antibiotic resistance by targeting the SOS response.
SOS 反应是一种细菌 DNA 损伤反应途径，它与细菌对抗生素产生耐药性的能力密切相关。SOS 反应的激活依赖于两种细菌蛋白 RecA 和 LexA 的相互作用。RecA 通过 ATP 依赖性方式在单链 DNA（ssDNA）上形成长的寡聚体纤维（RecA*），充当 DNA 损伤传感器。RecA* 然后可以结合 LexA，LexA 是 SOS 反应基因的抑制剂，触发 LexA 降解，从而诱导 SOS 反应。因此，RecA*-LexA 复合物的形成充当了关键的“SOS 激活信号”。由于研究涉及多种大分子相互作用的复杂反应存在挑战，因此，允许 LexA 切割的 RecA的基本成分尚未得到很好的定义。在这里，我们利用头对头连接和端封 RecA 构建体作为工具来定义能够与 LexA 结合的最小 RecA纤维。与之前提出的模型相反，我们发现仅三个连接的 RecA 单元就能够结合 ssDNA、LexA 和切割 LexA。我们进一步证明，RecA 寡聚化本身不足以切割 LexA，必需与 ATP 和 ssDNA 结合才能与连接的构建体形成有效的 SOS 激活信号。我们的 RecA*最小系统突出了先前 SOS 激活信号模型的局限性，并提供了一种新的工具，可以通过靶向 SOS 反应来减缓获得性抗生素耐药性的发展。
