Department of Biochemistry, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157-1016, USA.
Nucleic Acids Res. 2010 May;38(9):2865-77. doi: 10.1093/nar/gkp1245. Epub 2010 Jan 13.
Human chromosomal fragile sites are specific loci that are especially susceptible to DNA breakage following conditions of partial replication stress. They often are found in genes involved in tumorigenesis and map to over half of all known cancer-specific recurrent translocation breakpoints. While their molecular basis remains elusive, most fragile DNAs contain AT-rich flexibility islands predicted to form stable secondary structures. To understand the mechanism of fragile site instability, we examined the contribution of secondary structure formation to breakage at FRA16B. Here, we show that FRA16B forms an alternative DNA structure in vitro. During replication in human cells, FRA16B exhibited reduced replication efficiency and expansions and deletions, depending on replication orientation and distance from the origin. Furthermore, the examination of a FRA16B replication fork template demonstrated that the majority of the constructs contained DNA polymerase paused within the FRA16B sequence, and among the molecules, which completed DNA synthesis, 81% of them underwent fork reversal. These results strongly suggest that the secondary-structure-forming ability of FRA16B contributes to its fragility by stalling DNA replication, and this mechanism may be shared among other fragile DNAs.
人类染色体脆性位点是特定的位置，在部分复制应激条件下，这些位置特别容易发生 DNA 断裂。它们通常存在于参与肿瘤发生的基因中，并且映射到所有已知的癌症特异性重现性易位断点的一半以上。尽管它们的分子基础仍然难以捉摸，但大多数脆性 DNA 包含富含 AT 的灵活性岛屿，这些岛屿预计会形成稳定的二级结构。为了了解脆性位点不稳定性的机制，我们研究了二级结构形成对 FRA16B 断裂的贡献。在这里，我们表明 FRA16B 在体外形成了一种替代 DNA 结构。在人类细胞的复制过程中，FRA16B 表现出复制效率降低，并且取决于复制方向和与起始点的距离，会发生扩展和缺失。此外，对 FRA16B 复制叉模板的检查表明，大多数构建体在 FRA16B 序列内包含 DNA 聚合酶暂停，并且在完成 DNA 合成的分子中，81%的分子经历了叉反转。这些结果强烈表明，FRA16B 的二级结构形成能力通过阻止 DNA 复制导致其脆性，并且这种机制可能在其他脆性 DNA 中共享。
