The properties of Msh2-Msh6 ATP binding mutants suggest a signal amplification mechanism in DNA mismatch repair.
The properties of Msh2-Msh6 ATP binding mutants suggest a signal amplification mechanism in DNA mismatch repair.
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DOI：
10.1074/jbc.ra118.005439
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发表时间：
2018-11-23
期刊：
影响因子：
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通讯作者：
Kolodner RD
中科院分区：
文献类型：
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作者：
Graham WJ 5th;Putnam CD;Kolodner RD
DNA错配修复（MMR）纠正由DNA复制错误产生的错配DNA碱基以及小的插入/缺失环。在结合一个错配后，真核生物错配识别复合物Msh2 - Msh6在其两个核苷酸结合位点结合ATP，这诱导一种构象变化，导致形成一个Msh2 - Msh6滑动夹，它从错配处释放并沿着DNA自由滑动。然而，Msh2 - Msh6滑动夹在MMR中所起的作用仍知之甚少。在此，我们利用酿酒酵母（Saccharomyces cerevisiae）构建了Msh2和Msh6的Walker A核苷酸结合位点突变体，这些突变体在Msh2 - Msh6异二聚体的一个或两个核苷酸结合位点存在ATP结合缺陷。我们发现这些突变在体内导致完全的MMR缺陷。突变的Msh2 - Msh6复合物表现出正常的错配识别能力，并且能够熟练地招募MMR内切核酸酶Mlh1 - Pms1到错配的DNA上。在生理（2.5 mM）ATP浓度下，突变复合物在体外重组的不依赖Mlh1 - Pms1和依赖Mlh1 - Pms1的MMR反应中以及在激活Mlh1 - Pms1内切核酸酶方面表现出适度的部分缺陷，并且在低（0.1 mM）ATP浓度下表现出更严重的缺陷。相比之下，在高和低ATP浓度下，其中五个突变体在滑动夹形成方面完全有缺陷，一个则大部分有缺陷。这些发现表明，错配依赖的滑动夹形成触发额外的Msh2 - Msh6复合物的结合，并且在MMR过程中错配结合的信号放大需要进一步招募额外的下游MMR蛋白。
DNA mismatch repair (MMR) corrects mispaired DNA bases and small insertion/deletion loops generated by DNA replication errors. After binding a mispair, the eukaryotic mispair recognition complex Msh2–Msh6 binds ATP in both of its nucleotide-binding sites, which induces a conformational change resulting in the formation of an Msh2–Msh6 sliding clamp that releases from the mispair and slides freely along the DNA. However, the roles that Msh2–Msh6 sliding clamps play in MMR remain poorly understood. Here, using Saccharomyces cerevisiae, we created Msh2 and Msh6 Walker A nucleotide–binding site mutants that have defects in ATP binding in one or both nucleotide-binding sites of the Msh2–Msh6 heterodimer. We found that these mutations cause a complete MMR defect in vivo. The mutant Msh2–Msh6 complexes exhibited normal mispair recognition and were proficient at recruiting the MMR endonuclease Mlh1–Pms1 to mispaired DNA. At physiological (2.5 mm) ATP concentration, the mutant complexes displayed modest partial defects in supporting MMR in reconstituted Mlh1–Pms1-independent and Mlh1–Pms1-dependent MMR reactions in vitro and in activation of the Mlh1–Pms1 endonuclease and showed a more severe defect at low (0.1 mm) ATP concentration. In contrast, five of the mutants were completely defective and one was mostly defective for sliding clamp formation at high and low ATP concentrations. These findings suggest that mispair-dependent sliding clamp formation triggers binding of additional Msh2–Msh6 complexes and that further recruitment of additional downstream MMR proteins is required for signal amplification of mispair binding during MMR.