Department of Chemistry, Lomonosov Moscow State University, Leninskye Gory 1, Moscow 119991, Russia.
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Leninskye Gory 1, Moscow 119991, Russia.
Int J Mol Sci. 2023 Jan 5;24(2):1061. doi: 10.3390/ijms24021061.
G-quadruplexes (G4s), the most widely studied alternative DNA structures, are implicated in the regulation of the key cellular processes. In recent years, their involvement in DNA repair machinery has become the subject of intense research. Here, we evaluated the effect of G4 on the prokaryotic DNA mismatch repair (MMR) pathway from two bacterial sources with different mismatch repair mechanisms. The G4 folding, which competes with the maintenance of double-stranded DNA, is known to be controlled by numerous opposing factors. To overcome the kinetic barrier of G4 formation, we stabilized a parallel G4 formed by the d(GGGT) sequence in a DNA plasmid lacking a fragment complementary to the G4 motif. Unlike commonly used isolated G4 structures, our plasmid with an embedded stable G4 structure contained elements, such as a MutH cleavage site, required to initiate the repair process. G4 formation in the designed construct was confirmed by Taq polymerase stop assay and dimethyl sulfate probing. The G4-carrying plasmid, together with control ones (lacking a looped area or containing unstructured d(GT) insert instead of the G4 motif), were used as new type models to answer the question of whether G4 formation interferes with DNA cleavage as a basic function of MMR.
四链体（G4s）是研究最广泛的替代 DNA 结构之一，它们参与了关键细胞过程的调节。近年来，它们在 DNA 修复机制中的作用成为了研究的热点。在这里，我们评估了来自两种具有不同错配修复机制的细菌来源的 G4 对原核 DNA 错配修复（MMR）途径的影响。众所周知，G4 的折叠与双链 DNA 的维持相竞争，受许多相反因素的控制。为了克服 G4 形成的动力学障碍，我们稳定了一个在没有与 G4 基序互补片段的 DNA 质粒中形成的平行 G4。与常用的分离 G4 结构不同，我们带有嵌入式稳定 G4 结构的质粒包含了启动修复过程所需的元件，如 MutH 切割位点。通过 Taq 聚合酶停止测定和二甲磺酸探测证实了设计构建体中的 G4 形成。携带 G4 的质粒与对照质粒（缺乏环状区域或含有无结构的 d(GT)插入物而不是 G4 基序）一起被用作新的模型来回答 G4 形成是否干扰作为 MMR 基本功能的 DNA 切割的问题。
