Structural adaptation of vertebrate endonuclease G for 5-hydroxymethylcytosine recognition and function

DNA碱基编码的信息和碱基的各种化学修饰定义了不同类型的生物体。该项目的主要目标是确定脊椎动物特有的DNA修饰如何被脊椎动物（包括人类）特有的蛋白质所利用，以帮助修复受损的DNA。从这项研究中获得的知识将提高对蛋白质氨基酸序列在进化过程中看似微小的变化如何影响其结构的理解，并导致脊椎动物和无脊椎动物如何读取和利用其dna中的修饰的巨大差异。此外，这个项目将联合生物化学和艺术系共同开发一门新课程“科学中的视觉传达”。本课程将让科学和艺术的学生在一个协作的环境中学习如何以视觉上有影响力、科学上准确和信息丰富的方式为各种受众呈现科学概念和数据。5-羟甲基胞嘧啶（5hmC）是一种表观遗传修饰，在脊椎动物中具有多种功能，但在无脊椎动物中没有已知的功能。该项目旨在确定小鼠内切酶G （mEndoG）如何在四链Holliday连接的背景下切割5hmC位点的DNA以促进位点特异性重组。特定氨基酸的作用将通过在mEndoG和蠕虫同源物之间交换序列来研究，该同源物不会切割5hmc修饰的DNA。所得到的酶将通过x射线晶体学进行结构表征，并通过DNA结合和裂解试验检查其功能，以测试序列交换是否会导致脊椎动物和无脊椎动物同源物之间结构和功能的相应变化。最后，将研究mEndoG的连接识别，以确定该酶是否是5hmc特异性的连接分解酶，或者连接是否模拟另一种底物，例如DNA进入和离开染色质纤维中的核小体颗粒的交叉结构。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

The information encoded in DNA bases and various chemical modifications of the bases define different types of living organisms. The primary goal of this project is to determine how a DNA modification specific to vertebrate animals is used by a protein also found specifically in vertebrates, including humans, to help repair damaged DNA. The knowledge gained from this study will improve understanding of how seemingly small changes in the amino acid sequence of a protein through evolution can affect its structure and result in dramatic differences in how vertebrate versus invertebrate animals read and utilize modifications in their DNAs. In addition, this project will bring together biochemistry and art faculty to develop a new course on “Visual Communication in Science.” This course will engage science and art students in a collaborative environment to learn how to present scientific concepts and data in ways that are visually impactful and scientifically accurate and informative for a variety of audiences.5-Hydroxymethylcytosine (5hmC) is an epigenetic modification that serves multiple functions in vertebrates but has no known function in invertebrates. The project aims to determine how mouse endonuclease G (mEndoG) cleaves DNA at 5hmC sites in the context of four-stranded Holliday junctions to promote site-specific recombination. The roles of specific amino acids will be studied by swapping sequences between mEndoG and a worm homolog that does not cleave 5hmC-modified DNA. The resulting enzymes will be structurally characterized by X-ray crystallography and their functions examined by DNA binding and cleavage assays to test whether the sequence swaps result in corresponding changes in structures and functions between the vertebrate and invertebrate homologs. Finally, junction recognition by mEndoG will be studied to determine whether this enzyme is a 5hmC-specific junction resolvase or whether junctions mimic another substrate, such as the cross-over structures of DNA entering and exiting a nucleosome particle in chromatin fibers.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.