Structural role of 5-hydroxymethylcytosine in promoting recombination

提案标题：5-羟甲基胞嘧啶在促进重组中的结构作用众所周知，DNA的C、G、A和T的线性排列决定了细胞的遗传密码。我们现在还认识到，除了这种线性序列之外，DNA碱基的修饰也可以从亲本转移到子代细胞中，从而影响子代细胞如何解释这种密码;这就是表观遗传密码。甲基化是一种研究充分的表观遗传标记，但胞嘧啶上的甲基氧化形成羟甲基胞嘧啶（5 hmC）构成了一种不太常见但同样重要的表观遗传标记。该项目旨在确定5 hmC如何在原子水平上影响DNA的三维结构，影响重组，遗传信息在真核细胞（包括人类细胞）的染色体之间、病毒与其宿主之间转移的过程，以及修复受损的DNA。该项目的研究结果将对我们理解表观遗传标记及其生物学功能产生重要影响。潜在的经济影响是，这种理解将允许设计新技术，通过促进更有效的重组机制将基因引入生物系统。 此外，该项目将通过将实践经验带到当地高中的课堂，并将高中生带到科罗拉多州立大学校园，影响科学素养和高中生在STEM领域的培训。一个多样化的研究生骨干也将在STEM领域接受培训。 5-羟甲基胞嘧啶（5 hmC）作为基因组的“第六”碱基，最近又引起了人们的兴趣。5 hmC的功能通常被认为是5-甲基胞嘧啶去甲基化的中间体;然而，最近在基因组中对5 hmC的定位表明，修饰的碱基作为表观遗传标记，并以组织依赖性方式影响转录。此外，有证据表明，5 hmC通过依赖于通用核苷酸切割酶内切核酸酶G（EndoG）的途径促进体外和体内重组。该项目将测试5 hmC通过稳定重组中间体（四链DNA霍利迪连接）发挥结构作用的假设，该中间体可以为EndoG提供特异性，即使该酶似乎不能区分5 hmC修饰的DNA和未修饰的DNA。该项目的目标是测试5 hmC在反向重复序列中稳定Holliday连接的模型，从而为EndoG结合和切割提供稳定的中间体。目的：1）通过晶体学和量热学研究确定5 hmC修饰对DNA霍利迪连接结构和热力学稳定性的影响; 2）通过生物化学研究确定EndoG识别DNA中5 hmC修饰的结构背景和机制;和目的3）生物化学研究以确定5 hmC修饰是否暂停连接以允许EndoG进行位点特异性切割。

Proposal Title: Structural role of 5-hydroxymethylcytosine in promoting recombinationIt is well recognized that the linear arrangement of C, G, A, and T of DNA defines the genetic code of a cell. We also now recognize that, beyond this linear sequence, modifications to the DNA bases can also be transferred from the parent to affect how this code is interpreted in daughter cells; this is the epigenetic code. Methylation is a well-studied epigenetic marker, but the oxidation of methyl groups on cytosine, to form hydroxymethylcytosine (5hmC) constitutes a less common, but no less important epigenetic marker. This project aims to determine how 5hmC affects the three-dimensional structure of DNA at the atomic level, to affect recombination,The process by which genetic information is transferred between chromosomes in eukaryotic cells (including human cells), between a virus and its host, and to repair damaged DNAs. The results from this project will have significant impact on our understanding of epigenetic markers and their biological functions. The potential economic impact is that this understanding will allow the design of new technologies to introduce genes into biological systems by promoting more efficient mechanisms for recombination. The project will, in addition, impact the science literacy and the training of high school students in the STEM areas by bringing hands-on experiences to the classroom of local high schools and bringing high school students onto the Colorado State University campus. A diverse cadre of graduate students will also be trained in a STEM field. 5-Hydroxymethylcytosine (5hmC) has recently seen renewed interests as the "6th" base of the genome. The function of 5hmC has generally been recognized as being an intermediate in the demethylation of 5-methylcytosine; however, recent mapping of 5hmC occurrence across genomes suggests that the modified base serves as an epigenetic marker and affects transcription in a tissue dependent manner. In addition, evidence suggests that 5hmC promotes recombination in vitro and in vivo, through a pathway dependent on the general nucleotide cutting enzyme endonuclease G (EndoG). The project will test the hypothesis that 5hmC plays a structural role by stabilizing a recombination intermediate (the four stranded DNA Holliday junction), which can provide specificity to EndoG, even though the enzyme does not appear to discriminate between 5hmC-modified and unmodified DNAs. The goal of this project is to test a model that 5hmC stabilizes Holliday junctions in inverted repeat sequences, thereby providing a stable intermediate for EndoG to bind and cut. The aims to achieve this goal are: Aim 1) crystallographic and calorimetric studies to determine the effects of 5hmC-modification on the structure and thermodynamic stability of DNA Holliday junctions; Aim 2) biochemical studies to determine the structural context and mechanism by which EndoG recognizes 5hmC-modifications in DNA; and Aim 3) biochemical studies to determine whether 5hmC-modifications pauses the junction to allow site specific cutting by EndoG.