Plotz Guido, Piiper Albrecht, Wormek Marc, Zeuzem Stefan, Raedle Jochen
University of the Saarland, Klinik für Innere Medizin II, Homburg/Saar, Germany.
Biochem Biophys Res Commun. 2006 Feb 17;340(3):852-9. doi: 10.1016/j.bbrc.2005.12.096. Epub 2005 Dec 27.
Human DNA mismatch repair is initiated by MutSalpha which ATP-dependently recruits MutLalpha. Analysis of this complex is difficult due to its transient and dynamic nature. We have optimized conditions for investigation of MutLalpha.MutSalpha complexes using a DNA pulldown assay. Non-specific DNA end-binding, which frequently interfered with analysis of the interaction, did not occur under the applied conditions. MutSalpha had significantly higher affinity to DNA mispairs, but its interaction with MutLalpha did not require a mismatch. Complex formation was best supported by low magnesium concentration and low temperature at physiological pH and salt concentration. Complex formation was delayed by the slowly hydrolyzable ATP analog ATPgammaS, undetectable with the non-hydrolyzable analog AMP-PNP, and occurred weakly with a combination of AMP-PNP and ADP, confirming that hydrolysis was required. The described conditions likely capture an intermediate of the repair reaction which has bound ATP and ADP in the two nucleotide-binding sites of MutSalpha.
人类DNA错配修复由MutSα启动，MutSα以ATP依赖的方式招募MutLα。由于该复合物具有瞬时性和动态性，对其进行分析较为困难。我们利用DNA下拉实验优化了研究MutLα-MutSα复合物的条件。在所应用的条件下，经常干扰相互作用分析的非特异性DNA末端结合并未发生。MutSα对DNA错配具有显著更高的亲和力，但其与MutLα的相互作用并不需要错配。在生理pH值和盐浓度下，低镁浓度和低温最有利于复合物的形成。复合物的形成被缓慢水解的ATP类似物ATPγS延迟，用不可水解的类似物AMP-PNP无法检测到，而在AMP-PNP和ADP的组合下形成较弱，这证实了水解是必需的。所描述的条件可能捕获了修复反应的一个中间体，该中间体在MutSα的两个核苷酸结合位点中结合了ATP和ADP。
