Cruciform DNA structure underlies the etiology for palindrome-mediated human chromosomal translocations
Cruciform DNA structure underlies the etiology for palindrome-mediated human chromosomal translocations
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DOI：
10.1074/jbc.m400354200
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发表时间：
2004-08-20
影响因子：
4.8
通讯作者：
Toda, T
中科院分区：
文献类型：
--
作者：
Kurahashi, H;Inagaki, H;Toda, T
越来越多的证据表明，富含回文的AT重复序列(PATRR)代表了导致人类反复染色体易位的双链断裂热点。作为这种重排的一种机制，我们提出PATRR形成一个十字形结构，这是基因组不稳定的来源。为了验证这一假设，我们研究了克隆的PATRR的三级结构。我们已经观察到含有这种PATRR的质粒经历了构象变化，在琼脂糖凝胶电泳法上引起了依赖于温度的迁移率变化。在生理性盐浓度下观察到迁移率的变化，当在电泳法之前将质粒DNA在室温下孵育时，迁移率变化最为显著。双向凝胶电泳法分析表明，迁移率的变化是由于形成了十字形结构。S1核酸酶和T7核酸内切酶都将质粒切割成线形，也表明形成了十字形。此外，抗十字DNA抗体降低了含有PATRR的片段的电泳率。最后，我们用原子力显微镜直接显示了预期发夹臂大小的质粒DNA的十字形挤出。我们的数据表明，对人类染色体来说，易位易感性是由PATRRs介导的，可能是由于其不稳定的构象所致。
There is accumulating evidence to suggest that palindromic AT-rich repeats (PATRRs) represent hot spots of double-strand breakage that lead to recurrent chromosomal translocations in humans. As a mechanism for such rearrangements, we proposed that the PATRR forms a cruciform structure that is the source of genomic instability. To test this hypothesis, we have investigated the tertiary structure of a cloned PATRR. We have observed that a plasmid containing this PATRR undergoes a conformational change, causing temperature-dependent mobility changes upon agarose gel electrophoresis. The mobility shift is observed in physiologic salt concentrations and is most prominent when the plasmid DNA is incubated at room temperature prior to electrophoresis. Analysis using two-dimensional gel electrophoresis indicates that the mobility shift results from the formation of a cruciform structure. S1 nuclease and T7 endonuclease both cut the plasmid into a linear form, also suggesting cruciform formation. Furthermore, anti-cruciform DNA antibody reduces the electrophoretic mobility of the PATRR-containing fragment. Finally, we have directly visualized cruciform extrusions from the plasmid DNA with the size expected of hairpin arms using atomic force microscopy. Our data imply that for human chromosomes, translocation susceptibility is mediated by PATRRs and likely results from their unstable conformation.