Hydroxymethylcytosine Stabilizes Holliday Junctions and Promotes Recombination via Interaction with Endonuclease G

 描述（由申请人提供）：表观遗传修饰剂在指导DNA结构、调节基因表达和确定疾病状态方面发挥重要作用。科学家们最近对一种表观遗传标记5-羟甲基胞嘧啶（5 hmC）产生了兴趣，它在哺乳动物基因组中占胞嘧啶的不到1%。最初，5 hmC主要被认为是甲基胞嘧啶代谢的中间体，但现在该领域开始认识到5 hmC参与多种细胞功能，甚至称其为基因组的“第六个碱基”。一个观察结果是5 hmC的高局部基因组浓度与DNA重组有关。此外，最近发现重组蛋白内切酶G（Endo G）相对于未修饰的DNA优先切割5 hmC修饰的DNA。这项研究旨在探索5 hmC、Endo G和重组之间的联系。假设是5 hmC修饰的DNA形成独特的氢键，促进迁移的DNA霍利迪连接的暂停，并且暂停的连接为Endo G结合提供了稳定的底物。该模型将从结构角度进行探索，具有两个具体目标。第一个目的是使用X射线晶体学和差示扫描量热法确定5 hmC DNA的结构和热力学。第二个目的是通过测试Endo G与一系列5 hmC、5甲基-C和未修饰的-C双链体和连接DNA构建体的结合来确定5 hmC对Endo G识别的作用。总的来说，重组背后的机制尚未完全了解，5 hmC可能是调控过程的重要组成部分。由于重组是DNA损伤修复机制的一个组成部分，5 hmC可能有助于靶向局部DNA区域进行修复。特定的细胞类型，包括癌细胞，表达较低水平的5 hmC。这表明5 hmC水平与癌细胞的疾病状态有关，通过解除对DNA修复的调节。这项研究将有助于建立理解重组相关疾病的基本知识基础。对5 hmC的全面了解将进一步有助于解码基因组调控机制，影响不仅仅是重组领域。

 DESCRIPTION (provided by applicant): Epigenetic modifiers play vital roles in directing DNA structure, regulating gene expression, and determining disease-states. Scientists have recently taken interest in a epigenetic marker, 5-hydroxymethylcytosine (5hmC), which represents less than one percent of cytosines in the mammalian genome. Initially, 5hmC was thought primarily to be an intermediate in methylcytosine metabolism, but now the field is beginning to recognize 5hmC's involvement in a variety of cellular functions, even calling it the "sixth base" of the genome. One observation is that high local genomic concentrations of 5hmC have been linked to DNA recombination. Furthermore, the recombination protein Endonuclease G (Endo G) has been recently found to preferentially cleave 5hmC modified DNA over unmodified DNA. The proposed research aims to explore this link between 5hmC, Endo G, and recombination. The hypothesis is that 5hmC-modified DNA forms unique hydrogen bonds that promote pausing of a migrating DNA Holliday junction, and the paused junction provides a stable substrate for Endo G to bind. This model will be explored from a structural perspective with two specific aims. The first aim is to determine the structure and thermodynamics of 5hmC DNA using x-ray crystallography and differential scanning calorimetry. The second aim is to define the role of 5hmC on Endo G recognition by testing the binding of Endo G to a series of 5hmC, 5methyl-C, and unmodified-C duplex and junction DNA constructs. Overall, the mechanisms behind recombination aren't fully understood, and 5hmC is likely an important part the regulation process. Since recombination is an integral part of the DNA damage repair mechanism, 5hmC may help to target local DNA regions for repair. Particular cell types, including cancer cells, express lower levels of 5hmC. This suggests 5hmC levels are linked to the disease-state of cancer cells by de-regulating their DNA repair. This research will help build the fundamental base of knowledge for understanding recombination-related diseases. A complete understanding of 5hmC will furthermore help decode the mechanisms of genomic regulation, impacting more than just the recombination field.