上图最初用于说明三核苷酸重复序列扩增的分子机制。这方面公认的机制是与串联重复序列的动态突变相关的“DNA滑移复制”(Slippage DNA Replication)。我们在2021发表的文章中赋予滑移复制机制与重复链接的特定阈值的相关性。我们认为这种机制与原核和真核基因组中大量"短"( Short)串联重复序列(tandem repeats)的动态突变(Dynamic Mutation)有关。当通过增加重复长度打破这个“阈值”时，三核苷酸重复的不稳定机制将立即转变为其他类型的突变，并进入不稳定突变模式(Mutation Mode, 突变阈值）。当这个阈值也被跨越时，三核苷酸重复序列的不稳定性将主要取决于它们形成非-B DNA构象的能力。随着形成非-B结构的能力增加，这种非-B DNA结构造成的双链断裂修复的概率也增加。事实上，当前已有很多人提出了一些基于修复这些非-B结构引起的DNA双断裂的机制，其中大多数都有体外实验或模型系统（如大肠杆菌、酵母等）数据的支持或部分支持。

  在此基础上，结合特定DNA片段基因转录形成RNA:DNA杂交（又名R-loop）的现象，我们提出了一种可能的三核苷酸扩增机制，即R-loop介导的额外DNA复制（见上图）。这一机制于2006年首次提出，并在2014年发表的另一篇文章中给予强化。目前，在后生真核生物基因组DNA复制起始位点的“探究性”研究方面取得了一些进展。这些进展表明，G-四联体（G-quadruplex）或CpG岛(CpG islands)位点(后生生物基因转录的一种表观调控手段)和其他可形成R-环的DNA序列部位常常有与人类真核生物基因组中DNA复制位点的“许可”(Origin Lisencing 有关（ori识别蛋白ORC1与R-loop共同出现在这些区域）。

   这使得我早在2006年提出的上述机制的可信度进一步提高。希望随着时间的推移，能够对长期困扰医学和医学分子生物学界的50多种不同的人类神经肌肉疾病的病理机制将得到充分理解。

   The above figures were originally used to illustrate the molecular mechanism of trinucleotide repeats expansion. The well-recognized mechanism in this regard is the "slippage DNA replication" related to the dynamic mutation of the tandem repeats. In our article published in 2021, the slippage DNA replication is proposed to be a common mechanism of the tandem repeats replication that generates insertion and deletion instabilities when the repeat links are below a specific threshold. We believe that this mechanism is mainly related to the dynamic mutation of a large number of short tandem repeats in both prokaryotic and eukaryotic genomes. When this "threshold" is broken by increasing the repeat length by slippage DNA replication, the instability of long trinucleotide repeats will turn to use other types of mechanisms that is suggested to be involved in the interference of DNA repair with the repeats replication leading to the point mutation mode of instabilities (Mutation threshold). When this threshold is also crossed, the instability of the trinucleotide repeats will be mainly subject to the abnormal repair on the non-B DNA structures folded by long trinucleotide repeats that own significant capacities of forming non-B DNA conformation. As the chance of forming non-B structures increases, the repair of such non-B DNA structures is also required due to their effects on DNA metabolism. Indeed, some mechanisms based on the abnormal repair of DNA double strand breaks caused by cutting the non-B structures have been proposed, and most of them are supported or partially supported by the experiments carried out in vitro or in vivo using the model systems, such as E.coli, yeast, etc. In addition, we proposed a possible expansion mechanism of trinucleotide repeats underlying more than 50 human neuromuscular diseases, based on the fact that gene transcriptions through certain DNA segments of gene generate  RNA: DNA hybrid (aka R-loop), the mechanism has been given a name, additional DNA replication mechanism mediated by R-loop processing (see the above figures). This mechanism was first proposed in 2006 and was reinforced by another article published in 2014. In the same  time, some progresses have been made in the determination" of DNA replication initiation sites in eukaryotic genomes. It was shown that the sites where G- quadruplex (G-tetrad) potentially form or the CpG island regulating gene transcription epigenetically and other DNA sequences that can form R-loop are  related to the "licensing" of the sites of replication initiations  in human and other eukaryotic genomes (ORC1 recognition).

This gives the credibility of the above mechanism. It is hoped that over time, the pathological mechanism underlying more than 50 different human neuromuscular diseases will be fully understood.

附加信息：

真核生物基因组DNA复制起始位点的特征汇总

1、 在果蝇和小鼠等后生生物基因组中，绝大多数的CpG岛（CGI，CpG Islands）附近含有DNA复制起始位点Ori。尽管果蝇中几乎不存在甲基化，但大多数CpG岛（CGI）都含有ORI，这表明这种表观遗传学标记对于定义激活起源并不重要。DNA合成起始于CGI的边界，导致新生链呈明显的双峰分布，暗示了双重起始事件。

2、 富含GC的重复序列元件可用于后生生物基因组复制起点（Oris）的良好预测因子，表明后生生物基因组DNA复制起点也体现部分一致的碱基排列特征。

3、 通过标记新合成的DNA新生链发现，Ori处含有与新生链峰一致的独特核苷酸偏好，其特征是Ori位点5'和3'的G/T和C/A过度性存在。

4、 全基因组分析与DNA梳理分析相结合发现基因组中含有大量的可作为ORI的组织结构。但是，这些ORI的激活并不随机发生。它们被组织成特定位点，灵活使用ORI，可推测，这些Ori确定了一个复制子（Replicon），在每个复制子中激活(Firing)一个Ori。这样的组织使得每个复制子具有位点特异性和Ori firing的灵活性，可能与环境和细胞命运不同适应有关。

5、 小鼠胚胎干细胞中85%的复制起始位点与转录单位有关。近一半的已鉴定ORI图谱位于启动子区域，有趣的是，ORI密度与启动子密度密切相关，反映了小鼠基因组中复制和转录的协调组织。

6、 对ORI活性的详细分析表明，CpG岛启动子-ORI是胚胎干细胞（ES）中最有效的ORI，并且ORI特异性和点火（Firing）效率在不同细胞类型中都保持不变。而且启动子-ORI处复制起始位点的分布与转录起始位点（TSS）的分布完全平行，表明驱动复制和转录的调控区域共同进化。

7、 RNA：DNA杂交体的形成区和DNA复制起始位点的相关性。R-环与ORC共同定位在同一个CpG岛区域。这些有效的DNA复制起始位点的很大一部分正好位于G4（四链体）基序密度最高的位置。这种情景暗示人类细胞内基因转录和DNA复制之间的联系，并表明CpG岛启动子-DNA复制起始位点处形成R-环诱发的DNA复制失调可能与癌症细胞出现的DNA复制异常和基因组完整性缺失有关。

 参考文献

1、Wen Yale, Lü Kenao, Xu Xiaokang， Zhang Xin，Ding Liang, Pan Xuefeng  SMARCAL1, roles and mechanisms in genome stability maintenance. Hereditas(Beijing) ›› 2019, Vol. 41 ›› Issue (12): 1084-1098.doi: 10.16288/j.yczz.19-158

2、Xuefeng Pan, Nan Jiang, Xifang Chen, Xiaohong Zhou, Liang Ding, Fei Duan  R-loop structure: the formation and the effects on genomic stability. Hereditas (Beijing) 2014, 36 (12): 1185-94

3、Xuefeng Pan Mechanism of trinucleotide repeats instabilities: the necessities of repeat non-B secondary structure formation and the roles of cellular trans-acting factors. Acta Genetica SInica/Journal of Genetics and Genomics. 2006 Jan;33(1):1-11. doi: 10.1016/S0379-4172(06)60001-2. PMID: 16450581

4、Jin-Pu Wei, Xuefeng Pan, Hongquan Li, Fei Duan Distribution and evolution of simple repeats in the mtDNA D-loop in mammalian Hereditas(Beijing). 2011 Jan;33(1):67-74.  doi: 10.3724/sp.j.1005.2011.00067.

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