Schmidt K H, Abbott C M, Leach D R
Institute of Cell and Molecular Biology, University of Edinburgh, King's Buildings, Edinburgh EH9 3JR, UK.
Mol Microbiol. 2000 Jan;35(2):463-71. doi: 10.1046/j.1365-2958.2000.01727.x.
The expansion of normally polymorphic CTG microsatellites in certain human genes has been identified as the causative mutation of a number of hereditary neurological disorders, including Huntington's disease and myotonic dystrophy. Here, we have investigated the effect of methyl-directed mismatch repair (MMR) on the stability of a (CTG)43 repeat in Escherichia coli over 140 generations and find two opposing effects. In contrast to orientation-dependent repeat instability in wild-type E. coli and yeast, we observed no orientation dependence in MMR- E. coli cells and suggest that, for the repeat that we have studied, orientation dependence in wild-type cells is mainly caused by functional mismatch repair genes. Our results imply that slipped structures are generated during replication, causing single triplet expansions and contractions in MMR- cells, because they are left unrepaired. On the other hand, we find that the repair of such slipped structures by the MMR system can go awry, resulting in large contractions. We show that these mutS-dependent contractions arise preferentially when the CTG sequence is encoded by the lagging strand. The nature of this orientation dependence argues that the small slipped structures that are recognized by the MMR system are formed primarily on the lagging strand of the replication fork. It also suggests that, in the presence of functional MMR, removal of 3 bp slipped structures causes the formation of larger contractions that are probably the result of secondary structure formation by the CTG sequence. We rationalize the opposing effects of MMR on repeat tract stability with a model that accounts for CTG repeat instability and loss of orientation dependence in MMR- cells. Our work resolves a contradiction between opposing claims in the literature of both stabilizing and destabilizing effects of MMR on CTG repeat instability in E. coli.
某些人类基因中正常多态性CTG微卫星的扩增已被确定为包括亨廷顿舞蹈症和强直性肌营养不良在内的多种遗传性神经疾病的致病突变。在此，我们研究了甲基定向错配修复（MMR）对大肠杆菌中（CTG）43重复序列在140代以上稳定性的影响，并发现了两种相反的效应。与野生型大肠杆菌和酵母中依赖方向的重复序列不稳定性相反，我们在MMR缺陷的大肠杆菌细胞中未观察到方向依赖性，并提出对于我们所研究的重复序列，野生型细胞中的方向依赖性主要由功能性错配修复基因引起。我们的结果表明，在复制过程中会产生滑移结构，导致MMR缺陷细胞中单三联体的扩增和收缩，因为它们未得到修复。另一方面，我们发现MMR系统对这种滑移结构的修复可能会出错，导致大的收缩。我们表明，当CTG序列由后随链编码时，这些依赖mutS的收缩优先出现。这种方向依赖性的本质表明，MMR系统识别的小滑移结构主要在复制叉的后随链上形成。这也表明，在存在功能性MMR的情况下，去除3个碱基的滑移结构会导致形成更大的收缩，这可能是CTG序列二级结构形成的结果。我们用一个解释CTG重复序列不稳定性和MMR缺陷细胞中方向依赖性丧失的模型，对MMR对重复序列稳定性的相反效应进行了合理化解释。我们的工作解决了文献中关于MMR对大肠杆菌中CTG重复序列不稳定性的稳定和不稳定效应的相反观点之间的矛盾。
