Abstract

The mismatch repair system (MMR) ensures the stability of genetic information during DNA replication in almost all organisms. Mismatch repair is initiated after recognition of a non-canonical nucleotide pair by the MutS protein and the formation of a complex between MutS and MutL. Eukaryotic and most bacterial MutL homologs function as endonucleases that introduce a single-strand break in the daughter strand of the DNA, thus activating the repair process. However, many aspects of the functioning of this protein remain unknown. We studied the ATPase and DNA binding functions of the MutL protein from the pathogenic bacterium Neisseria gonorrhoeae (NgoMutL), which exhibits endonuclease activity. For the first time, the kinetic parameters of ATP hydrolysis by the full-length NgoMutL protein were determined. Its interactions with single- and double-stranded DNA fragments of various lengths were studied. NgoMutL was shown to be able to efficiently form complexes with DNA fragments that are longer than 40 nucleotides. Using modified DNA duplexes harboring a 2-pyridyldisulfide group on linkers of various lengths, we obtained NgoMutL conjugates with DNA for the first time. According to these results, the Cys residues of the wild-type protein are located at a distance of approximately 18–50 Å from the duplex. The efficiency of the affinity modification of Cys residues in NgoMutL with reactive DNAs was shown to decrease in the presence of ATP or its non-hydrolyzable analog, as well as ZnCl₂, in the reaction mixture. We hypothesize that the conserved Cys residues of the C-terminal domain of NgoMutL, which are responsible for the coordination of metal ions in the active center of the protein, are involved in its interaction with DNA. This information may be useful in reconstruction of the main stages of MMR in prokaryotes that are different from γ-proteobacteria, as well as in the search for new targets for drugs against N. gonorrhoeae.

中文翻译：

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淋病奈瑟菌错配修复系统的MutL蛋白：与ATP和DNA的相互作用

摘要

错配修复系统（MMR）可确保几乎所有生物体在DNA复制过程中遗传信息的稳定性。MutS蛋白识别出非经典核苷酸对并在MutS和MutL之间形成复合物后，就会启动错配修复。真核生物和大多数细菌的MutL同源物起核酸内切酶的作用，在DNA的子链中引入单链断裂，从而激活了修复过程。然而，该蛋白功能的许多方面仍然未知。我们研究了来自淋病奈瑟氏球菌的MutL蛋白的ATPase和DNA结合功能（NgoMutL），具有核酸内切酶活性。首次确定了全长NgoMutL蛋白水解ATP的动力学参数。研究了其与各种长度的单链和双链DNA片段的相互作用。NgoMutL被证明能够与长度超过40个核苷酸的DNA片段有效地形成复合物。使用修饰的DNA双链体在各种长度的接头上都带有一个2-吡啶基二硫键基团，我们首次获得了与DNA结合的NgoMutL缀合物。根据这些结果，野生型蛋白的Cys残基距离双链体约18–50Å。NgoMutL中的Cys残基与反应性DNA亲和修饰的效率显示在ATP或其不可水解类似物以及ZnCl存在下会降低₂，在反应混合物中。我们假设NgoMutL的C端结构域的保守Cys残基参与了蛋白质活性中心中金属离子的配位，参与了它与DNA的相互作用。该信息可能有助于重建不同于γ-变形杆菌的原核生物中MMR的主要阶段，以及寻找针对淋病奈瑟氏球菌的新靶标。