Bourn Rebecka L, Rindler Paul M, Pollard Laura M, Bidichandani Sanjay I
Department of Biochemistry & Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.
Mutat Res. 2009 Feb 10;661(1-2):71-7. doi: 10.1016/j.mrfmmm.2008.11.003. Epub 2008 Nov 13.
Expanded triplet repeat sequences are known to cause at least 16 inherited neuromuscular diseases. In addition to short-length changes, expanded triplet repeat tracts frequently undergo large changes, often amounting to hundreds of base-pairs. Such changes might occur when template or primer slipping creates insertion/deletion loops (IDLs), which are normally repaired by the mismatch repair system (MMR). However, in prokaryotes and eukaryotes, MMR promotes large changes in the length of (CTG.CAG)(n) sequences, the motif most commonly associated with human disease. We tested the effect of MMR on instability of the expanded (GAA.TTC)(n) sequence, which causes Friedreich ataxia, by comparing repeat instability in wild-type and MMR-deficient strains of Escherichia coli. As expected, the prevalence of small mutations increased in the MMR-deficient strains. However, the prevalence of large contractions increased in the MMR mutants specifically when GAA was the lagging strand template, the orientation in which replication fork stalling is known to occur. After hydroxyurea-induced stalling, both orientations of replication showed significantly more large contractions in MMR mutants than in the wild-type, suggesting that fork stalling may be responsible for the large contractions. Deficiency of MMR promoted large contractions independently of RecA status, a known determinant of (GAA.TTC)(n) instability. These data suggest that two independent mechanisms act in response to replication stalling to prevent instability of the (GAA.TTC)(n) sequence in E. coli, when GAA serves as the lagging strand template: one that is dependent on RecA-mediated restart of stalled forks, and another that is dependent on MMR-mediated repair of IDLs. While MMR destabilizes the (CTG.CAG)(n) sequence, it is involved in stabilization of the (GAA.TTC)(n) sequence. The role of MMR in triplet repeat instability therefore depends on the repeat sequence and the orientation of replication.
已知扩展的三联体重复序列会导致至少16种遗传性神经肌肉疾病。除了短片段变化外，扩展的三联体重复序列经常会发生大幅变化，通常可达数百个碱基对。当模板或引物滑动产生插入/缺失环（IDL）时，可能会发生这种变化，而IDL通常由错配修复系统（MMR）修复。然而，在原核生物和真核生物中，MMR会促进（CTG.CAG）（n）序列长度的大幅变化，该基序与人类疾病最为相关。我们通过比较野生型和MMR缺陷型大肠杆菌菌株中的重复序列不稳定性，测试了MMR对导致弗里德赖希共济失调的扩展（GAA.TTC）（n）序列不稳定性的影响。正如预期的那样，MMR缺陷型菌株中小突变的发生率增加。然而，特别是当GAA作为后随链模板时，MMR突变体中大幅收缩的发生率增加，已知复制叉在此方向会发生停滞。在羟基脲诱导停滞之后，MMR突变体中复制的两个方向都比野生型显示出明显更多的大幅收缩，这表明复制叉停滞可能是大幅收缩的原因。MMR缺陷促进了大幅收缩，且与RecA状态无关，RecA状态是（GAA.TTC）（n）不稳定性的已知决定因素。这些数据表明，当GAA作为后随链模板时，有两种独立的机制响应复制停滞，以防止大肠杆菌中（GAA.TTC）（n）序列的不稳定性：一种依赖于RecA介导的停滞叉的重新启动，另一种依赖于MMR介导的IDL修复。虽然MMR会使（CTG.CAG）（n）序列不稳定，但它参与了（GAA.TTC）（n）序列的稳定。因此，MMR在三联体重复序列不稳定性中的作用取决于重复序列和复制方向。
