Institute of Molecular Cancer Research, University of Zürich, Zürich, Switzerland.
Nat Struct Mol Biol. 2013 Apr;20(4):486-94. doi: 10.1038/nsmb.2520. Epub 2013 Mar 3.
Expansion of GAA/TTC repeats is the causative event in Friedreich's ataxia. GAA repeats have been shown to hinder replication in model systems, but the mechanisms of replication interference and expansion in human cells remained elusive. To study in vivo replication structures at GAA repeats, we designed a new plasmid-based system that permits the analysis of human replication intermediates by two-dimensional gel electrophoresis and EM. We found that replication forks transiently pause and reverse at long GAA/TTC tracts in both orientations. Furthermore, we identified replication-associated intramolecular junctions, located between GAA/TTC repeats and other homopurine-homopyrimidine tracts, that were associated with breakage of the plasmid fork not traversing the repeats. Finally, we detected postreplicative, sister-chromatid hemicatenanes on control plasmids, which were replaced by persistent homology-driven junctions at GAA/TTC repeats. These data prove that GAA/TTC tracts interfere with replication in humans and implicate postreplicative mechanisms in trinucleotide repeat expansion.
GAA/TTC 重复扩展是弗里德里希共济失调的致病事件。研究表明，GAA 重复在模型系统中会阻碍复制，但人类细胞中复制干扰和扩展的机制仍不清楚。为了研究 GAA 重复处的体内复制结构，我们设计了一种新的基于质粒的系统，该系统允许通过二维凝胶电泳和 EM 分析人类复制中间体。我们发现，复制叉在两种方向上的长 GAA/TTC 片段处都会短暂暂停和反转。此外，我们还鉴定了与未穿过重复序列的质粒叉断裂相关的复制相关的分子内连接，这些连接位于 GAA/TTC 重复和其他同聚嘌呤-同聚嘧啶片段之间。最后，我们在对照质粒上检测到复制后姐妹染色单体半随体，这些半随体在 GAA/TTC 重复处被持续的同源驱动连接所取代。这些数据证明 GAA/TTC 片段在人类中干扰复制，并暗示复制后机制在三核苷酸重复扩展中起作用。
