MutS recognition: Multiple mismatches and sequence context effects
MutS recognition: Multiple mismatches and sequence context effects
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DOI：
10.1007/bf02704758
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发表时间：
2001-12-01
影响因子：
2.9
通讯作者：
Rao, BJ
中科院分区：
文献类型：
--
作者：
Joshi, A;Rao, BJ
大肠杆菌MutS是一种多功能修复蛋白，不仅特异性识别各种类型的错配，而且还识别长度高达4个核苷酸的单链环。特异性结合，随后是追踪定位半甲基化位点的DNA螺旋的下一步，由蛋白质的构象状态作为ATP结合/水解的函数来调节。在这里，我们研究了异源双链的各种分子决定因素如何调节错配识别MutS，错配修复的关键第一步。使用经典的DNA酶I足迹分析，我们证明了MutS结合到各种类型的错配的层次结构是相同的，无论错配是单独存在还是多重存在。此外，这种独特的层次结构是漠不关心的差异水平的DNA螺旋的灵活性和不配对的状态的错配碱基在异源双链体。令人惊讶的是，与类似的单一错配相比，多个错配表现出降低的与MutS结合的亲和力。这种亲和力的降低可能是由于序列背景效应，我们通过研究改变的序列背景中的两个相同的单一错配更直接地建立了这种效应。错配，在简单地被翻转在相同的位置，eliminates MutS特异性接触的变化，从而强调序列上下文在调节MutS结合到错配的重要性。
Escherichia coli MutS is a versatile repair protein that specifically recognizes not only various types of mismatches but also single stranded loops of up to 4 nucleotides in length. Specific binding, followed by the next step of tracking the DNA helix that locates hemi-methylated sites, is regulated by the conformational state of the protein as a function of ATP binding/hydrolysis. Here, we study how various molecular determinants of a heteroduplex regulate mismatch recognition by MutS, the critical first step of mismatch repair. Using classical DNase I footprinting assays, we demonstrate that the hierarchy of MutS binding to various types of mismatches is identical whether the mismatches are present singly or in multiples. Moreover, this unique hierarchy is indifferent both to the differential level of DNA helical flexibility and to the unpaired status of the mismatched bases in a heteroduplex. Surprisingly, multiple mismatches exhibit reduced affinity of binding to MutS, compared to that of a similar single mismatch. Such a reduction in the affinity might be due to sequence context effects, which we established more directly by studying two identical single mismatches in an altered sequence background. A mismatch, upon simply being flipped at the same location, elicits changes in MutS specific contacts, thereby underscoring the importance of sequence context in modulating MutS binding to mismatches.