As no crystal structure of full-size MutL bound to DNA has been obtained up to date, in the present work we used crosslinking and Förster resonance energy transfer (FRET) assays for probing the putative DNA-binding center of MutL from Escherichia coli. Several single-cysteine MutL variants (scMutL) were used for site-specific crosslinking or fluorophore modification. The crosslinking efficiency between scMutL proteins and mismatched DNA modified with thiol-reactive probes correlated with the distances from the Cys residues to the DNA calculated from a model of MutS-MutL-DNA complex. FRET-based investigation of DNA binding with different scMutL variants clearly showed that the highest signals were detected for the variants MutL(T218C) and MutL(A251C) indicating closeness of the positions 218 and 251 to DNA in the MutL-DNA complex. Indeed, the Cys218 and Cys251 of scMutL were crosslinked to the reactive DNA with the highest yield demonstrating their proximity to DNA in the MutL-DNA complex. The presence of MutS increased the yield of conjugate formation between the MutL variants and the modified DNA due to tighter MutL-DNA interactions caused by MutS binding to MutL.

中文翻译：

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通过交联和福斯特共振能量转移，从大肠杆菌错配修复系统中探寻MutL蛋白的DNA结合中心。

由于迄今尚未获得与DNA结合的全尺寸MutL的晶体结构，因此在本工作中，我们使用交联和Förster共振能量转移（FRET）分析方法从大肠杆菌中探究了MutL的假定DNA结合中心。几种单半胱氨酸MutL变体（scMutL）用于位点特异性交联或荧光团修饰。scMutL蛋白与硫醇反应探针修饰的错配DNA之间的交联效率与从Muts-MutL-DNA复合物模型计算出的Cys残基到DNA的距离有关。基于FRET的DNA与不同scMutL变体结合的研究清楚地表明，检测到变体MutL（T218C）和MutL（A251C）的信号最高，表明MutL-DNA复合物中DNA的位置218和251接近。确实，scMutL的Cys218和Cys251以最高的产率与反应性DNA交联，表明它们与MutL-DNA复合物中的DNA接近。MutS的存在增加了MutL变体与修饰的DNA之间共轭物形成的产率，这是由于MutS与MutL的结合导致MutL-DNA相互作用更紧密所致。